1

FINAL PROGRAM – ABSTRACT BOOK

European Association of

Tissue Banks http://www.eatb2013.eu/

2

Edited by J.-P. Pirnay. The results published in this book of abstracts are under the full responsibility of the authors.

3

Table of content

Welcome to the 2013 EATB meeting in Brussels!..................................................... 4

General information…………………………………………………………………………………………….. 5

Organisation……………………………………………………………………………………………………….…9

Networking events……………………………………………………………………………………………….10

Sponsors and exhibitors……………………………………………………………………………………….11

Scientific information…………………………………………………………………………………………..12

Interactive workshops………………………………………………………………………………………….13

Floor plans congress venue….……………………………….……………………………………………..14

Program at a glance……………………………………………………………………………………………..17

Scientific program………………………………………………………………………………………………..18

Short bio’s of convenors and invited speakers……………………………………………………..29

Abstracts of oral presentations…………………………………………………………………………….78

Abstracts of poster presentations……………………………………………………...……………….160

Author index……………………………………………………………………………………………………….220

4

Welcome to the 2013 EATB meeting in Brussels!

Dear Friends and Colleagues, We take great pleasure in welcoming you to the International Congress of the EATB in Brussels. This 22nd EATB Congress is organized as a joint meeting with the Belgian Transplantation Society (BTS), the Belgian Transplant Coordination Organization (BTCO), the Belgian Association of Cell and Tissue Banks (BACTB) and the European Normalizing Institution (CEN). Belgium is the warmhearted country of beer, chocolate and comic strips. Its capital city Brussels has a central location in the heart of Europe (European Commission and Council of the European Union), and because it has been Burgundian, Spanish, Austrian, French and Dutch throughout time, visitors will find a diversity of beauty: art, architecture and of course gastronomy. The congress venue is the Royal Military Academy (RMA), which is situated in the central part of the capital and next to the European Institutions. The 2013 EATB conference provides a good opportunity to present and discuss the latest developments in the cell and tissue banking field, from research to clinical practice and including some “burning” topics, such as Ethical Issues, Legislation, Vigilance and Surveillance, ATMPs, Tissue Engineering and Cell Therapy. In addition, also this year the popular interactive workshops on “Donor selection” (10th edition!) and “Quality Assurance” will be organized. We are delighted with the interest in this conference: more than 130 abstracts were submitted by delegates from 23 countries. About 80 abstracts were selected for oral presentation and about 50 for poster presentation. We are thankful to all contributions as well as to the numerous companies and governmental agencies that sponsored this meeting. We also wish to thank the many individuals, including the members of the Local Organising and Scientific Committees, who are dedicated to making this meeting successful. We invite you to participate in the scientific program, the networking reception at the Brussels City Hall and the gala dinner at the Solvay Library. On behalf of the European Association of Tissue Banks we wish you a pleasant stay and hope you will find this meeting intellectually and socially rewarding.

Ramadan Jashari, MD, FETCS President of the EATB

Jean-Paul Pirnay, MScEng, PhD Chair of the Scientific Committee

5

General information

Meeting venue

The Royal Military Academy The conference building

The Royal Military Academy is a military institution of university education responsible for the basic academic, military and physical training of future officers, and for the continuing advanced training of officers during their active career in the Defence department. The officers graduated from the Academy are leaders capable of performing efficiently in diverse, complex and exceptional circumstances, at the service of the national and the international community. The training at the Academy is tailored to the needs of the Belgian Defence (army, air force, navy, medical service). The values of society are integrated into the formation.

Entrance address of the conference centre:

Hobbemastraat 8, 1000 Brussels.

How to reach the Royal Military Academy Nearby subway stations:

There are two subway stations at 10-15 minutes walk from the Royal Military Academy: Schuman & Merode (Line 1 and 5). Coming from Brussels Grand Place:

The Central station is located at walking distance; there you can take subway line 1 or 5 to the stations Schuman or Merode.

6

How to walk from Schuman subway station (green flag) to the Royal Military Academy (red

flag):

At the Schuman roundabout take the Rue de La loi up into the direction of the park and carry on for 275m. Turn left at Avenue de la Joyeuse Entrée and carry on for 285m. Turn right at Avenue de la Renaissance and carry on for 350m. Turn left at Rue Hobbema-street and carry on for 200m to reach the entrance of the Royal Military Academy.

How to walk from Merode subway station (green flag) to the Royal Military Academy (red

flag)?

At the roundabout avenue de Tervuren, turn into avenue de l’Yser and carry on for 600m. Then turn right at rue Hobbema and carry on for 200m to reach the entrance of the Royal Military Academy.

7

Opening hours registration Tuesday, Nov. 19: 10:00 – 18:00 Wednesday, Nov. 20: 08:30 – 18:00 Thursday, Nov. 21: 08:00 – 18:00 Friday, Nov. 22: 08:30 – 09:00

Badges Participants should collect name badges from the conference registration desk (in the conference building). You are kindly asked to show your badge when entering the Royal Military Academy and to wear your badge in the conference building. The first time you enter the Royal Military Academy (without badge) you will need to register

at the access control point (this will take only a few minutes and you will be assisted by

congress staff).

Certificate of attendance The certificates of attendance are issued at the registration desk. You will receive it at the same time as you register or pick up your registration material.

Cloakroom and storage facility A cloakroom is available in the conference building (in between the two registration desks).

Note that the conference building, and thus also the cloakroom, closes for the night

immediately after the end of the scientific program.

The patio area (poster viewing, catering and exhibition) does not close for the night. Exhibitors can store small materials (laptops, gadgets, brochures,..) in a dedicated room in the patio area. This room will be closed at 18:30.

Coffee and lunch breaks During the session breaks coffee or lunch will be served (buffet) free of charge to participants wearing badges. Coffee and lunch breaks will take place in the patio of the building left of the conference building. During the two interactive workshops (“Donor selection” and Quality assurance”), lunch will

also be served at the workshop site (studio 1/2 in the basement of the conference building).

Currency The official currency of Belgium is the Euro (€).

8

Drinking water The drinking water in Belgium is of good quality and can be used without concern.

Internet

Free Internet access will be available in the conference building and in the patio area.

Language The official language of the congress is English.

Message board A message board is available in the registration area.

Program changes The conference organisers reserve the right to modify the program and cannot assume any liability for changes in the program due to external or unforeseen circumstances.

Staff Should you have any questions, congress staff will be pleased to help you. Please contact the registration desk.

9

Organisation

Organising Committee

Congress President

Ramadan Jashari, MD, FETCS Members

Hilde Beele, MD, PhD Pierre Neirinckx, Col, MD, SBH Chris Deroubaix, Lt Col, MAB Bruno Pascual, MSc Eng Bruno Deschans, MSc Jean-Paul Pirnay, MSc Eng, PhD Nadine Ectors, MD, PhD Caroline Van Geyt, MSc Ye Dong Fan, MD, PhD Gilbert Verbeken, MSc Johan Guns, MSc Harry Vindevogel, Maj Gen, MSc Eng Ludo Muylle, MD, PhD

Scientific Committee

Chair

Jean-Paul Pirnay, MSc Eng, PhD Members

Etienne Baudoux, Belgium John Kearney, UK Hilde Beele, Belgium Ján Koller, Slovakia Arlinke Bokhorst, The Netherlands Johan Kurz, Austria Martin Börgel, Germany Peter Lodewyckx, Belgium Scott Brubaker, USA Torsten Malm, Sweden Carl Ceulemans, Belgium Dimitri Mikhalski, Belgium Jill Davies, UK Hans-Joachim Mönig, Germany Theo De By, The Netherlands Aurora Navarro Martinez-Cantullera, Spain Daniel De Vos, Belgium Richard Ngakam Noumanje, Belgium Timothy Devos, Belgium Robert Parker, UK Jean-Pierre Draye, Belgium Axel Pruss, Germany Denis Dufrane, Belgium Thomas Rose, Belgium Ted Eastlund, USA Johan Somville, Belgium Nadine Ectors, Belgium Jaroslav Spatenka, Czech Republic Patrick Evrard, Belgium Esteve Trias, Spain Ye Dong Fan, Belgium Izabela Uhrynowska-Tyszkiewicz, Poland Deirdre Fehily, Italy Ivan Van Riet, Belgium Simone Hennerbichler-Lugscheider, Austria Alain Vanderkelen, Belgium Isabelle Huys, Belgium Gilbert Verbeken, Belgium Ramadan Jashari, Belgium Ruth Warwick-Cohen, UK Artur Kaminski, Poland

10

Networking events Wednesday November 20, 20:00 – 23:00

Networking Reception

Participants: free

Accompanying persons: Euro 30

Thursday November 21, 20:00 – 24:00

Gala Dinner

Participants: Euro 50

Accompanying persons: Euro 70

The networking reception of the EATB 2013 congress will be organized in the Brussels City Hall, located on the famous Grand Place in Brussels.

A masterpiece of gothic civil architecture from the 15th century, the Brussels City Hall is also famous for the richness of its interior. Paintings, sculptures and tapestries evoke local and national history in one of the most sumptuous environments in the country.

In the Gothic building, two ancient rooms were reorganized in the 19

th century by the architect of the City, Victor Jamaer.

This was done in a pure neogothic style. One of them, the ‘Gothic Room,’ kept its role for official receptions. It is in this room that the networking reception will be held.

Upon registration you will receive a voucher.

This is a great opportunity to meet your fellow conference attendees.

Situated in Leopold Park in the heart of the European Quarter, the Solvay Library is an ideal location for an exclusive dinner event.

Behind its simple classical facade, the Solvay Library hides a rich decor of precious wood, mosaics and stained glass windows. This listed building, designed in 1902 by famous architects Constant Bosmans and Henri Vandeveld, is one of the finest examples of eclecticism in Belgium.

Its construction was financed by the Belgian industrialist Ernest Solvay. The building was formerly home to the Institute for Sociology of the Université Libre de Bruxelles.

The ground and first floors are built around a magnificent Reading Room with study rooms and galleries to either side. The lower ground floor has been renovated in a contemporary style and is ideal for gala dinners.

A quartet from the Royal Band of the Belgian Navy will enrich the evening with classical music.

Admission only for registered participants! Registration is

mandatory in order to receive a voucher.

11

Sponsors and Exhibitors

SILVER SPONSOR

SPONSORS

Centro Nazionale Trapianti Istituto Superiore di Sanità

12

Scientific information

Oral presentations

It is essential that you upload your presentations, preferably the morning before your talk. Staff members will be present in the registration area to upload and preview your presentation. Projection and technical setting

All rooms will be equipped with data-projection (no slides), using Windows 7 Professional SP 1 -64 Bits and Microsoft Office 2010.

The lecture rooms are exclusively equipped with Windows-PCs (no Macintosh machines). Mac-users are requested to bring their own notebook, particularly when using videos in the mov-format.

Please bring a USB-stick or CD-ROM formatted for Windows® (PC). You may want to carry a second stick/CD as a back-up in case there is any insoluble technical problem.

File Format: Microsoft® PowerPoint™ presentation formatted for Windows® (PC) only (Operating system: Windows 7 Professional SP 1 -64 Bits).

Please rehearse your talk to make sure it will fit comfortably into the available time.

Poster Presentations

Poster area

Platform in the patio of the building next to the conference building.

Presence at posters

Posters can be viewed during the entire meeting. In order to enable discussion and interaction with other participants, we request you (or one of your group) to be at your poster board for

the following presentation times:

Thursday, Nov. 21, 09:30 – 10:00 and 16:00 – 16:30 (Coffee breaks).

If this is not possible, please leave a note on your poster board detailing the times when you will be present at the board.

Format

The usable surface on the poster board will be 90 cm width x 130 cm height (approx. 35 x 51 inches), upright format (portrait).

Mounting and removal

Poster mounting Tuesday, Nov. 19: 10:00 – 18:00 Wednesday, Nov. 20: 08:30 – 10:00

Poster numbers will be posted on the poster boards and your board number will correspond to your abstract number in the program. Velcro tape will be provided.

Poster removal Friday, Nov. 22: 11:00 – 19:00

13

Interactive workshops

Workshop: Quality assurance Date and time: Thursday Nov. 21 from 12.00 to 14.00 Place: Studio 1/2, conference building – basement Chair: Deirdre Fehily, Italy

Cases are invited from anyone involved in tissue and cell banking and will be presented by the person submitting the case, their nominee or a Workshop Facilitator, and discussed by participants working in groups, facilitated by the above mentioned facilitators (practitioners with extensive experience of quality management in tissue and cell banks).

The cases considered will include examples where staff errors, equipment failure, system inadequacies or organisational difficulties resulted in Quality System deviations.

During the workshop, participants will discuss how each case should have been managed and what the appropriate corrective and preventive actions were. The case presenter will then share with the workshop the actual outcome of the case. The workshop will allow us to learn from each other how we should best manage unexpected incidents to ensure that recipient safety is not compromised and irreplaceable tissues and cells are not wasted!

Workshop: Donor selection Date and time: Friday Nov. 22 from 12.00 to 14.00 Place: Studio 1/2, conference building – basement Chair: Hilde Beele, Belgium

Cases are invited from anyone involved in tissue banking and will be presented by the person submitting the case, their nominee or one of the Workshop Facilitators.

The cases considered will include examples where donor medical history details or difficult ethical considerations made the decision about acceptance of the donor, or use of the tissue, challenging.

The participants are divided in small working groups, each of which is supported by a Workshop Facilitator (a practitioner with extensive experience of working with donors and their families), for discussion of the case.

The views of the working groups will be shared with the whole workshop and the actual outcome of each case will be reported. It is anticipated that the case discussions will subsequently be written up for publication on the EATB website or elsewhere.

Lunch will also be provided in Studio 1/2 during the workshop! The Chair and the facilitators of each workshop will select appropriate cases from the submissions to avoid duplication and allow for time constraints. You will be notified by email if your case has been selected for discussion.

14

Floor plans congress venue

The Royal Military Academy

15

Congress building – Ground floor

Congress building – First floor

Registratio

n D

esk C

loakro

om

Lecture R

oo

m

to S

tud

io 1

/2

(ba

sem

en

t)

Lecture H

all

16

Patio – Ground floor

Ent

ranc

e 1

�

Ent

ranc

e 2

�

Cat

eri

ng

Poster pla orm

1

3

2

8

4

7

6

5

Cat

eri

ng

1

4

11

10

13

12

9

1. ABN nv 2. Air Liquide Medical 3. AL.CHI.MI.A S.R.L. 4. Dr. Köhler Chemie GmbH 5. EMCM b.V. 6. Gebauer Medizintechnik 7. ICCBBA 8. EUROCET128 – Istituto

Superiore di Sanita – Centr Nazionale Trapianti

9. Macopharma 10. Mak-System 11. PMT Benelux nv 12. Scaldis St-Martin SA 13. Spierings Tissue Processing 14. Stem alpha SA 15. WAK Chemie medical GmbH

15

17

PROGRAM AT A GLANCE

Tuesday, November 19

10:00-18:00 REGISTRATION 10:00-18:00 MOUNTING OF EXHIBITION AND CATERING

Wednesday, November 20

08:30-18:00 REGISTRATION

08:30-10:00 Coffee and pastries

10:00-10:15 Welcome addresses

10:15-11:00 S01 – Ethical issues

11:00-12:00 S02 – Donation

12:00-14:00 Lunch 12:00-14:00 GENERAL ASSEMBLY

14:00-16:00 S03 – Regulatory affairs: perspectives of policymakers, competent authorities and practitioners

16:00-16:30 Coffee break / Poster viewing

16:30-18:30 S04 – Import / export and coding systems

20:00-23:00 NETWORKING RECEPTION AT BRUSSELS CITY HALL

Thursday, November 21

08:00-18:00 REGISTRATION

08:00-08:30 Coffee and pastries

08:30-09:30 S05 – Quality and safety 08:30-09:30 S06 – CEN / TC 316 forum

09:30-10:00 Coffee break / Poster presentation (presence of author is required)

10:00-12:00 S07 – Tissue processing Session in collaboration with the European Committee for Standardisation (CEN)

12:00-14:00 Lunch 12:00-14:00 S08 – Quality assurance workshop (lunch will also be served in studio 1/2)

14:00-16:00 S09a – Cell and tissue engineering / cell therapy I Session in collaboration with the Belgian Association of Tissue and Cell Banks

16:00-16:30 Coffee break / Poster presentation (presence of author is required)

16:30-18:00 S09b – Cell and tissue engineering / cell therapy II Session in collaboration with the Belgian Association of Tissue and Cell Banks

20:00-24:00 SOCIAL EVENT AND DINNER IN THE SOLVAY LIBRARY

Friday, November 22

08:30-09:00 REGISTRATION

08:30-09:00 Coffee and pastries

09:00-10:30 S10 – Transmissible infectious agents (incl. emerging infectious diseases)

10:30-11:00 Coffee break / Poster viewing

11:00-12:00 S11 – Biovigilance Rapid Alert for Tissues and Cells (RATC) / counterfeit - fraud

12:00-14:00 Lunch 12:00-14:00 S12: Donor selection workshop in collaboration with the Belgian Transplantation Society (lunch in studio 1/2)

14:00-15:30 S13a – Clinical application I 14:00-15:30 S14a – Clinical application of cardiovascular grafts I

15:30-16:00 Coffee break

16:00-17:00 S13b – Clinical application II 16:00-17:00 S14b – Clinical application of cardiovascular grafts II

17:00-17:30 Award ceremony / closing remarks / announcement EATB 2014

17:30-19:00 FAREWELL RECEPTION

Registration Desk Lecture Hall Patio Studio 1/2 Lecture Room

18

08:30 – 10:00 PATIO

Coffee and pastries

10:00 – 10:15 LECTURE HALL

Welcome addresses

R. Jashari, EATB president

Representative of the Royal Military Academy

10:15 – 11:00 LECTURE HALL

Session 1: Ethical issues

Chair: L. Noël, World Health Organisation, J.-P. Pirnay, Belgium, R. Jashari, Belgium

001 Truth and transparency in tissue donation S. Bartlett, Canada

002 Self-sufficiency and the non-commercial nature of tissues of human origin L. Noël, World Health Organisation

11:00 – 12:00 LECTURE HALL

Session 2: Donation

Chair: J. Davies, UK, C. Van Geyt, Belgium, P. Evrard, Belgium

003 Collaborating with medical examiners and coroners to increase donation M. Haun, Canada

004 Cornea donation and allocation in the Netherlands: the Dutch perspective H. A. van Leiden, The Netherlands

005 Cardiovascular tissue – extending donor age limits, UK experience J. Davies, UK

006 Validation of Oxford Quality Assessment Procedure to extend cardiovascular tissue donor age limits

J. Davies, UK

12:00 – 14:00

LECTURE HALL

GENERAL ASSEMBLY EATB

PATIO

Lunch

WEDNESDAY, NOVEMBER 20

PROGRAM

19

14:00 – 16:00 LECTURE HALL

Session 3: Regulatory affairs: perspectives of policy makers, competent authorities and

practitioners

Chair: N. Ectors, Belgium C. Chabannon, France, I. Huys, Belgium

007 Requirements for a clinical grade cellular cancer vaccine – what it takes to get through M. Eyrich, Germany

008 Consequences of the ATMP regulation in the field of Hematopoietic Stem Cell Transplantation

C. Chabannon, France

009 The EU regulatory framework for quality and safety of human tissues and cells S. Van der Spiegel, European Commission

010 The concept of « Placing on the market » under the EU regulatory framework: some flexibility for ATMPs? M. Pittie, Belgium

011 EU-wide economic overview of the markets of tissues and cells for transplantation T. de By, The Netherlands

012 Queen Astrid Military hospital’s 26-year old human keratinocyte cultures became Advanced Therapy Medicinal Products at the dawn of the third millennium: historical reflection and practical impact of this evolution

G. Verbeken, Belgium

16:00 – 16:30 PATIO

Coffee break / Poster viewing

16:30 – 18:30 LECTURE HALL

Session 4: Import / export and coding systems

Chair: R. Warwick, UK, J. Guns, Belgium, I. Siska, European Commission

013 Import – export and related hot topics S. Brubaker, US

014 Import – export of human tissues in the EU E. Trias, Spain

015 The emergence of global governance for Medical Products of Human Origin (MPHO) L. Noël, World Health Organisation

016 Information Standard for Blood and Transplant, ISBT 128, the globally endorsed standard for Medical Products of Human Origin (MPHO) R. Warwick, UK

017 Towards the implementation of the single European coding for human tissues and cells intended for human application I. Siska, European Commission

018 Getting ready for implementation of the single European code – information for tissue banks

D. Fehily, Italy

WEDNESDAY, NOVEMBER 20

PROGRAM

WEDNESDAY, NOVEMBER 20

PROGRAM

20

20:00 – 23:00 BRUSSELS CITY HALL

NETWORKING RECEPTION

WEDNESDAY, NOVEMBER 20

PROGRAM

21

08:00 – 08:30 PATIO

Coffee and pastries

08:30 – 09:30

LECTURE HALL

Session 5: Quality and safety

Chair: D. Fehily, Italy, G. Verbeken, Belgium, B. Golubovic, Croatia

019 Guide to the quality and safety of tissues and cells for human application – keeping the Council of Europe guidance up-to-date

M. López Fraga, Council of Europe

020 Safety, efficacy and quality: Balancing risks and benefits in ATMPs M. Hildebrandt, Germany

021 Cleanroom management in cell and tissue banking; the weakest link of the chain is the strongest, because it can break it J. Klykens, Belgium

022 Analysis of potential factors affecting microbiological cultures in tissue donors during procurement

C. Van Geyt, Belgium

LECTURE ROOM

Session 6: CEN/TC 316 forum

Chair: M. Harder, Germany, M. Doser, Germany

023 Standardization of tools for tissue engineering M. Doser, Germany

024 Biobanking: technologies and quality control A. Leichtle, Switzerland

025 Biobanking: regulatory issues and best practice M. Harder, Germany

09:30 – 10:00 PATIO

Coffee break / Poster presentation (presence of author is required!)

THURSDAY, NOVEMBER 21

PROGRAM

22

10:00 – 12:00 LECTURE HALL

Session 7 (in collaboration with the European Committee for Standardization (CEN)):

Tissue processing

Chair: M. Harder, Germany, A. Kaminski, Poland, E. Agustí, Spain

026 Banking of cardiovascular allografts: cryopreservation of conventional versus engineered tissues

R. Jashari, Belgium

027 Banking skin G. Verbeken, Belgium

028 Banking cornea: Today and tomorrow I. Martinache, France

029 Banking musculoskeletal tissue: which storage temperature is necessary? C. Fölsch, Germany

030 In vivo study on osteoconductivity of supercritical CO2 processed bone allografts, impregnated with tobramycin or vancomycin

D. P. Link, The Netherlands

031 Effect of gamma rays (G) and accelerated electron beam (EB) on compact bone collagen damage by assessment of collagen in vitro solubility: influence of defatting, radiation dose and irradiation temperature

A. Kaminski, Poland

032 MyStem Kit: a non-enzymatic closed system for minimal manipulation of adipose tissue for regenerative medicine applications

C. Cicione, Italy

033 Minimization of allogeneic tissue immunogenicity by cryopreservation K. G. M. Brockbank, US

034 Mesenchymal stem cell immunosuppressive therapy Y. Beguin, Belgium

12:00 – 14:00

STUDIO 1/2

Session 8: Quality assurance workshop Lunch will also be served in Studio 1/2!

Chair: D. Fehily, UK

PATIO

Lunch

THURSDAY, NOVEMBER 21

PROGRAM

23

14:00 – 16:00 LECTURE HALL

Session 9a (in collaboration with the Belgian Association of Tissue and Cell Banks):

Cell and tissue engineering / cell therapy I

Chair: T. Devos, Belgium, E. Trias, Spain, L. Muylle, Belgium

035 The Cardiac Atrial Appendage Stem Cell: a new candidate for myocardial repair J.-L. Rummens, Belgium

036 Limbal epithelial stem cell culture: an ongoing effort M.-J. Tassignon, Belgium

037 Qualitative and quantitative differences of adipose tissue-derived stem cells from superficial and deep subcutaneous lipoaspirates: a matter of fat

F. Michetti, Italy

038 Customized bone allografts for complex defect reconstruction: the use of computer numerically controlled milling

T. Schubert, Belgium

039 Comparison of different stem cell populations for vascularization and integration of an acellular human dermis graft seeded with autologous cells for standardized defect wounds

M. Vitacolonna, Germany

040 Tissue engineered biovital human skin substitute W. Łabuś, Poland

041 Regeneration of ischemic muscle with a composite graft made of adipose mesenchymal stem cells or bone marrow mesenchymal stem cells and human acellular collagen matrix

M. van Steenberghe, Belgium

042 Different methods of cell viability measurement applied on CBU-attached segments: a comparative study of their predictive value

T. Devos, Belgium

16:00 – 16:30 PATIO

Coffee break / Poster presentation (Presence of author is required!)

THURSDAY, NOVEMBER 21

PROGRAM

24

16:30 – 18:00 LECTURE HALL

Session 9b (in collaboration with the Belgian Association of Tissue and Cell Banks):

Cell and tissue engineering / cell therapy II

Chair: D. Dufrane, Belgium, S. Hennerbichler-Lugscheider, Austria, E. Baudoux, Belgium

043 Beta cell therapy for diabetes D. Pipeleers, Belgium

044 Active specific immunotherapy for High Grade Glioma S. Van Gool, Belgium

045 A 3-Dimensional osteogenic-like structure from human autologous adipose mesenchymal stem cells: Reproducibility, Genetic stability, clinical safety/efficacy D. Dufrane, Belgium

046 Development of a scaffold for human amniotic membrane handling and/or storage F. Gindraux, France

047 The use of high-definition optical coherence tomography and reflectance confocal microscopy to evaluate cellular and acellular human dermal matrices

J.-P. Draye, Belgium

048 Human mesenchymal stromal cells expanded in vitro in media supplemented with human platelet lysate

M. Skific, Croatia

049 Development of ATMPs from bench to patient: how to navigate the European Regulatory framework

P. Celis, European Medicines Agency

20:00 – 23:00 SOLVAY LIBRARY

SOCIAL EVENT AND DINNER

THURSDAY, NOVEMBER 21

PROGRAM

25

26

08:30 – 09:00 PATIO

Coffee and pastries

09:00 – 10:30

LECTURE HALL

Session 10: Transmissible infectious agents (incl. emerging infectious diseases)

Chair: T. L. Pitt, UK, D. De Vos, Belgium, Y.-D. Fan, Belgium

050 Antibiotic decontamination of banked tissues – a microbiologist’s viewpoint T.L. Pitt, UK

051 Emerging infectious disease outbreaks and the safety of tissues and cells transplantation in Europe

D. Domanovic, European Centre for Disease Prevention and Control

052 Towards a common microbiological testing protocol of human tissues and cells for transplantation?

V. Saegeman, Belgium

053 Optimalization of bone/tendon tissue procurements in term of microbiological contamination

V. Vandenschrik, Belgium

054 False negative results in microbiological analyses of corneal tissues A. Limongelli, Italy

055 Bacteriology testing of cardiovascular tissues: comparison of transport solution versus tissue testing in two different labs

R. Díaz Rodríguez, Belgium

10:30 – 11:00 PATIO

Coffee break / Poster viewing

11:00 – 12:00 LECTURE HALL

Session 11: Biovigilance / Rapid Alert for Tissues and Cells (RATC) / counterfeit - fraud

Chair: D. Fehily, Italy, I. Uhrynowska-Tyszkiewicz, Poland, A. Vanderkelen, Belgium

056 Tissue vigilance: lessons learned from the SOHO V&S project D. Fehily, Italy

057 Model of Cooperation between National Competent Authority (NCA’s) and Police F. Teskrat, France

058 Identificaton errors and double check procedures M. J. Happel, The Netherlands

FRIDAY, NOVEMBER 22

PROGRAM

27

12:00 – 14:00

STUDIO 1/2

Session 12 (icollaboration with the Belgian Transplant Society): Donor selection workshop Lunch will also be served in Studio 1/2!

Chair: H. Beele, Belgium

PATIO

Lunch

14:00 – 15:30

LECTURE HALL

Session 13a: Clinical application / follow up I

Chair: D. Ponzin, Italy, J. Somville, Belgium, T. Rose, Belgium

059 Clinical strategy of application of deep frozen – radiation sterilised bone allografts W. Marczyński, Poland

060 Endothelial cell transplantation on synthetic vascular bypasses J. Meinhart, Austria

061 Outcome of corneal transplantation D. Ponzin, Italy

062 Evaluation of human amniotic membrane associated with dermal substitute in the management of full thickness burns

A.-S. Hatzfeld, France

063 Acellular dermal matrix M. L. Pérez, Spain

064 Are CO2 cleaned allogenic bone grafts (eCOO-technology) as a high local dose antibiotic carrier system (OSTEOmycin) a safe and sufficient option for treatment of implant related infections and chronic osteomyelitis in a one-stage revision procedure?

K. Kaudela, Austria

065 Treatment of bone non-union by a composite graft made of concentrated bone marrow and demineralized bone matrix processed in a cleanroom S. Cuppens, Belgium

LECTURE ROOM

Session 14a: Clinical application / follow up of cardiovascular grafts I

Chair: R. Jashari, Belgium, J. Spatenka, Czech Republic, T. Malm, Sweden

066 Vascular homografts: life savers in transplantation and oncology surgery J. Lerut, Belgium

067 Development of global cardiovascular nomenclature I. Uhrynowska-Tyszkiewicz, Poland

068 Arterial allograft bypasses in critical limb ischemia: a thirteen-year experience H. Chenorhokian, France

FRIDAY, NOVEMBER 22

PROGRAM

28

069 Treatment of aortic valve endocarditis with homograft: indications and long term

outcome. G. El Khoury, Belgium

070 Pulmonary homografts for right ventricular outflow tract reconstruction during Ross procedure: twenty years results

A. Prat, France

15:30 – 16:00 PATIO

Coffee break

16:00 – 17:00

LECTURE HALL

Session 13b: Clinical application / follow up II

071 Full thickness skin grafts: quantifying graft contraction following explantation L. Jares, Spain

072 ‘Activated Bone’ a novel treatment approach for large bone defects, even in infections; a Case Report K. Kaudela, Austria

073 The use of human skin products in burn wound treatment T. Rose, Belgium

LECTURE ROOM

Session 14b: Clinical application / follow up of cardiovascular grafts II

074 Quality of life after the Ross procedure - reward for tissue bankers J. Spatenka, Czech Republic

075 RVOT replacement with homograft in congenital heart disease B. Meyns, Belgium

076 Current clinical experience with tissue-engineered allogenic matrices for pulmonary and aortic valve replacement S. Sarikouch, Germany

17:00 – 17:30 LECTURE HALL

Award ceremony / closing remarks / announcement EATB 2014

17:30 – 19:00 PATIO

FAREWELL RECEPTION

FRIDAY, NOVEMBER 22

PROGRAM

FRIDAY, NOVEMBER 22

PROGRAM

29

Short bio’s of convenors and invited speakers

Ramadan Jashari, 30 Nadine Ectors, 31 Hilde Beele, 32 Jean-Paul Pirnay, 33 Johan Guns, 34 Pierre Neirinckx, 35 Bruno Pascual, 36 Caroline Van Geyt, 37 Sandra Bartlett, 38 Luc Noël, 39 Matthias Eyrich, 40 Christian Chabannon, 41 Stefaan Van der Spiegel, 42 Marc Pittie, 43 Gilbert Verbeken, 44 Scott A. Brubaker, 45 Esteve Trias i Adroher, 46 Ruth Warwick, 47 Ioana Siska, 48 Deirdre F. Fehily, 49 Marta López Fraga, 50 Martin Oliver Hildebrandt, 51 Michael Doser, 52 Alexander Benedikt Leichtle, 53 Michael Harder, 54 Isabelle Martinache, 55 Christian Oliver Fölsch, 56 Kevin G. M. Brockbank, 57 Jean-Luc Rummens, 58 Marie-José Tassignon, 59 Daniel Pipeleers, 60 Stefaan Van Gool, 61 Denis Dufrane, 62 Patrick Celis, 63 Tyrone L. Pitt, 64 Dragoslav Domanovic, 65 Veroniek Saegeman, 66 Fewzi Teskrat, 67 Wojciech J. Marczyński, 68 Thomas Rose, 69 Diego Ponzin, 70 Jan Lerut, 71 Izabella Anna Uhrynowska-Tyszkiewicz, 72 Hovan Chenorhokian, 73 Gebrine El Khoury, 74 Alain Prat, 75 Bart Meyns, 76 Samir Sarikouch, 77

30

Ramadan Jashari Studies of medicine and surgery followed at the University of Rijeka and the University of Zagreb, Croatia and at the Catholic University of Leuven, Belgium. Training in cardiac surgery completed at the OLV Clinic in Aalst and at UZ Leuven, Belgium. He was a fellow of the EACTS in 2001 (FETCS). Currently he is the Medical Director of the European Homograft Bank (EHB) and a staff member cardiac surgeon at the Brussels Heart Centre (BHC), Clinic St. Jean in Brussels. He has been involved in the field of cardiovascular tissue banking as the responsible of research and development of the European Homograft Bank (EHB) from 1998 to 2007 and since 2008 he is Medical Director. He is a member of different scientific Associations: European Association of Cardio-Thoracic Surgery (EACTS), Belgian Association of Cardio-Thoracic Surgery (BACTS), Society of Heart Valve Disease (SHV) and European Association of Tissue Banks (EATB). He was a Board member of the BACTS from 2004 to 2011 and a Board member of the EATB between 2009 and 2012. Since November 2012 he is the President of the EATB. Dr. Jashari participated in 106 international and national scientific meetings and presented more than 75 abstracts and 13 posters. He published 36 articles in International Journals, whereof 24 in the field of tissue banking and clinical application of human tissues. He is currently member of the Editorial Board of the Scientific Journal “Cell and Tissue Banking” and a member of the ETTAG of the ISBT 128. He participated and participates currently in different European scientific projects, such as: “Do-He-Ca” (2013-2015), “ESPOIR” (2011-2014), “Euro-GTP” (2008-2011) and he has been involved in different studies in the field of cardio-vascular tissue engineering in Belgium, France and Sweden. In 2013 he was appointed a member of the CAT at the EMA.

31

Nadine Ectors Nadine Ectors is coordinator of the Activity Center biobanking of the University Hospitals Leuven since 2011. This activity center regroups most of the cell- and tissue banks of UZ Leuven as well as the diagnostic and scientific tissue collections used for scientific research. N. Ectors is professor at the Faculty of Medicine of the University of Leuven. She trained in medicine and specialized in pathology (The University of Leeds, UK; KU Leuven). Between 1994 and 2007, she was staff member of the department of pathology at UZ Leuven and between 2001 and 2011 director of cell and tissue banks at UZ Leuven. Additionally she obtained a PhD, a certificate in hospital governance and management, and quality management (KU Leuven). She is author and co-author of several peer-reviewed papers. N. Ectors is member of numerous scientific and professional organisations.

32

Hilde Beele Hilde Beele qualified from the Ghent University (Belgium) as a medical doctor in 1987. As a Research Assistant of the Belgian National Foundation for Scientific Research, she was able to gain experience in cell and tissue culture at the department of Histology of the Ghent University. In 1992, she graduated as Doctor in Biomedical Sciences at the Ghent University. In that period she was appointed as medical director of the centralised Tissue Bank of the Ghent University Hospital. The Tissue Bank of the Ghent University Hospital has accreditations for many types of cells and tissues: ophthalmic tissue; musculoskeletal tissue, vascular tissue, tympano-ossicular grafts, amniotic membrane and cultured keratinocytes. Nowadays, Hilde Beele is Head of Dept. of the Tissue Bank at the Ghent University Hospital. Meanwhile, she was trained as a dermatologist at the dept. of Dermatology of the Ghent University Hospital. In October 1993, she graduated as a dermatologist and became staff-member at the Department of Dermatology, where her main functions are to supervise the in-patients at the hospitalisation department and to run the ulcer clinic. Since a few years, H. Beele is Head of Dept. of the Wound Care Center, the multidisciplinary in- and out-patient ulcer clinic of the Ghent University Hospital. She is also president of EduWond, an initiative of Ghent University and its associated graduate schools, to promote training in wound healing. In 2000, H. Beele was appointed as Professor at the Ghent University. She is involved in the education of wound healing, tissue banking and regenerative medicine for nurses and students in medical and biomedical sciences. She is member/ board member of a number of advisory committees and scientific associations, both national and international. Since 2009, she is vice-president of the Superior Health Council, an advisory council of the Belgian Ministry of Health. Prof. dr. Hilde Beele, MD, PhD Dept. of Dermatology and Tissue Bank Ghent University Hospital De Pintelaan 185 B-9000 Gent BELGIUM Tel.: 09/332 33 81 Fax: 09/332 38 38 hilde.beele@ugent.be

33

Jean-Paul Pirnay Born in 1967 in Antwerp, Belgium. He graduated as Industrial Engineer in Biochemistry and Biotechnology at the University College Ghent and obtained a Scientific Degree in Agriculture Development at the University of Ghent. He received his PhD in Medical Sciences from the Vrije Universiteit Brussel. In 1993 he performed his military service, but did not walk away and served since then as a researcher in the Queen Astrid Military Hospital (QAMH) in Brussels, where he contributed to the development of the cell and tissue banks and to the introduction of molecular microbiology and bacteriophage therapy. Jean-Paul is currently head of the Laboratory for Molecular and Cellular Technology (LabMCT), which harbours the human cell and tissue banks of the QAMH. He is involved in several research projects, such as the development of living skin equivalents and bacteriophage cocktails for use in severely burnt patients, and several EU funded projects (FP7 COST Action BM1003, EuroGTP, FP7 PhagoBurn). He was recently appointed as Research Collaborator at the Royal Military Academy. He is also member of the Board and of the ‘Human Cell, Tissue and Organ’ Working Party of the Belgian Superior Health Council, a link between government policy and the scientific world in the field of public health in Belgium and participated in several National, EU (DG Sanco and DG Research) and NATO (Research Task Group HFM-194) evaluation and advisory committees. Jean-Paul has published about 45 peer reviewed journal articles and book chapters. His publications reflect his wide interests, from molecular microbiology to ethical issues. Outside his professional life, he loves painting and tries not to be the worst tennis player of Belgium.

Email: jean-paul.pirnay@mil.be

34

Johan Guns

Education: 1997: Bachelor of Pharmaceutical and Biological Techniques, Erasmus, Belgium 2000: Master of Medical and Social Science, Free University of Brussels, Belgium Professional Experience:

• 1997-2000: Research assistant, beta cell phenotyping, Free University of Brussels

• 2000-2007: Laboratory coordinator, Beta Cell Bank, University Hospital Brussels

• Since 2007: Quality manager Medical Laboratories and Tissue Banks, University Hospital Brussels

Scientific activities:

• Expert member of the working group “Cells, Tissues, and Organs of Human and Animal Origin” of the Belgian Superior Health Council

• Consultant expert Technical Committee PIC’s PE010-3, Belgian Cleanroom Workclub

• Consultant expert Technical Committee ISO/TC 209, Belgian Cleanroom Workclub Scientific associations:

• President of the Belgian Association of Tissue and Cell Banks (BVWB-ABBT)

• Member of the European Association of Tissue Banks (EATB)

• Member of the Belgian Society for Reproductive Medicine (BSRM)

35

Pierre Neirinckx Pierre Neirinckx was born on the 15th August 1962 in Namur, Belgium. He studied in the Royal Cadet School in Brussels. He graduated as a Doctor in Medicine at the Catholic University of Louvain (UCL) in 1987. He started his military career as General Practitioner in a Reconnaissance Battalion from 1987 to 1994. During this period he was involved in the medical training for Recon troops and Special Forces and was a regular guest-speaker in the NATO Long Range Recon Patrol School. As Medical Officer he deployed with his unit in several operations in all the spectrum of military interventions, as well in ROMANIA, as in the BALKANS, in RWANDA, in the DEMOCRATIC REPUBLIC of CONGO, in AFGHANISTAN and in LEBANON. In 1993 Capt Dr Neirinckx specialized in the Emergency Medicine and was part time member of the Emergency Department of the University Hospital of MONT-GODINNE. He was involved in several training courses for physicians, nurses and paramedics in emergency medicine and disaster management courses. In 2001, Maj Dr Neirinckx attended the higher General Staff course at the Royal Defence College in Brussels and graduated with a thesis entitled “Health Risk Assessment for Operations”. He was then appointed to the Staff of EUROCORPS in STRASBOURG (FRANCE). In 2004, he deployed as Deputy Medical Advisor of ISAF (AFGHANISTAN). In 2005, LTC Dr Neirinckx was Commanding Officer of a joint medical unit. In 2007 he commanded the BEL Field Hospital deployed in UNIFIL (LEBANON). From July 2009 up to now, Col Dr Neirinckx is the Director of the Military Hospital Queen Astrid in BRUSSELS. As a regular lecturer at the Catholic University of Leuven, he is still active in the education and training of civilian and military medical personnel in Disaster Medicine and Disaster Management. He completed the High Studies for Security and Defence at the Royal High Institute of Defence in 2013. Beside his national military activities, Col Dr Neirinckx is working for the Secretariat General of the International Committee of Military Medicine (ICMM) since 1999. In December 2011, he was elected by the General Assembly in the function of Deputy Secretary General.

36

Bruno Pascual

°02/04/1967 Brussels

Education:

- Msc, Industrial engineer in Biochemistry and Biotechnology (CTL Ghent) - Scientific degree in Agriculture development (University Ghent) Work experience:

1994-1998: Collaborator at travel agency Taxistop (department ’Home Exchange’) 1998-2005: Technician & research collaborator at Blood bank and Department of

Hematology of UZ Ghent 2006-2007: Projectcoordinator at Analytico nv (Wilrijk) (environmental lab for soil and waste

waters) 2008-today: Technician in skin and keratinocyte bank, Lab MCT, Burn Wound Centre of the

Queen Astrid Military hospital, Brussels.

37

Caroline Van Geyt Caroline Van Geyt is since 2005 staff member at the centralised tissue bank at the Ghent University Hospital. She is responsible for the coördination of the activities of the bank for amniotic membrane (for ophtalmologic indications), ocular tissue (sclera and cornea), keratinocytes (cell suspensions and sheets), muskoloskeletal tissues, vascular allografts and tympano-ossicular allografts, with the focus on continuous quality improvement. Before joining the tissue bank with a master degree of medical and social sciences, she worked in industry as project manager for the development of HBV diagnostic assays where she led the project from research, over development to (GMP-)production, through clinical trials and up till (post-) marketing phase. Previously, she also worked at Ghent University on a stem cell project elaborating isolating, cell culturing and cell typing techniques.

38

Sandra Bartlett Sandra Bartlett, Canada, is an award winning reporter and producer based in Toronto. She worked on the ICIJ project Secrecy for Sale and Skin and Bone. Bartlett worked as a producer and reporter in NPR's Investigative Unit based in Washington where she collaborated on projects with PBS Frontline, ProPublica, the Center for Public Integrity, the Center for Investigative Reporting, as well as individual journalists in Canada and Europe. Recent stories include Post Mortem, a series on the flaws in America's death investigation system, Poisoned Places, a look at whether the Clean Air Act has improved the air in America and Skin and Bone, an examination of the under-regulated business of recycling dead humans. In 20 plus years at the Canadian Broadcasting Corporation, as an editor, a reporter and producer, Bartlett covered daily news, foreign assignments and special programming. She worked in London, Europe, Israel, Cuba and Pakistan. Her investigative stories at CBC ranged from the dangerous prescribing of mood altering drugs in the elderly, to tax scams that cost the Canadian government hundreds of millions of dollars, an examination of police use of the Taser stun gun. This story included independent testing of Taser stun guns to reveal how little police knew about a weapon they used excessively. In 2007 Bartlett co-chaired the Global Investigative Journalism Conference held in Toronto. This conference brought 650 journalists from 30 countries to share and discuss investigative stories and techniques. The conference also created an international award, The Global Shining Light Award for journalists from developing countries or countries in transition who risk their lives for their stories.

39

Luc Noël Dr Noël received his medical degree from the University of Grenoble and specialized in clinical biology, haematology and transfusion medicine in Lyon and Paris. He worked as a French public hospital practitioner, in charge of the Haematology and Transfusion Centre of Versailles when he was recruited in 1992 to contribute to the establishment of the French Blood Agency following the tainted blood affair. He participated in the reorganization of the French Transfusion Service, contributed to setting up the system of vigilance and surveillance of adverse reactions, known as haemovigilance, and to optimizing the clinical use of blood components.- In 1999 he was recruited by WHO as Coordinator of Blood Transfusion Safety. - In 2004 he was appointed Coordinator for the Clinical Procedures (CPR) unit inter alia "in charge of promoting the appropriate effective and safe use of cell, tissue and organ transplantation, including surveillance of risks, in particular in xenotransplantation trials", following World Health Assembly Resolution WHA57-18. In 2010, after a global consultation process, the Assembly adopted Resolution WHA63.22 and endorsed the updated WHO Guiding Principles for Human Cell Tissue and Organ Transplantation.- In April 2013 Dr Noël was appointed Special Adviser in charge of a new Organization-wide initiative for medical products of human origin .

40

Matthias Eyrich DOB 04.08.1969

Professional training / activities:

July 1996 - December 1997 resident in pediatrics at the Children’s Hospital of the University of Tübingen

January 1998 - June 1999 postdoctoral fellowship: „T-cell reconstitution after haploidentical stem cell transplantation“

July 1999 - March 2004 completing residency in pediatrics at the Children’s Hospital of the University of Tübingen

April – August 2004 postdoctoral fellowship at Pasteur Institute, Paris, France

since Septembter 2004 head of stem cell processing laboratory at the University of Würzburg

since Mai 2006 appointment as Senior Consultant of the Stem Cell Transplant Unit at the Children’s Hospital Würzburg

March 2006 head of clinical laboratory for hematology and pediatric cytology

July 2006 habilitation in pediatrics

March 2007 subspecialisation in pediatric hematology/oncology

December 2009 endowed professorship for experimental and clinical cellular therapy (donated by the Parent’s Initiative Group for Children with Leukemia- and Solid Tumors Würzburg e.V.)

Current research activities:

➫ immune reconstitution after allogeneic stem cell transplantation in children

➫ stem cell differentiation into early committed T-cell progenitors (CLP-T)

➫ dendritic cell research and clinical scale DC production

➫ DC-vaccination trials in human high-grade gliomas

Member of the following societies:

1997 German Society for Pediatric and Adolescence Medicine (DGKJ)

1999 European Haematology Association (EHA)

2000 European Group for Bone Marrow Transplantation (EBMT)

2004 German society for Pediatric Oncology and Hematology (GPOH)

2009 International Society for Cellular Therapy (ISCT)

41

Christian Chabannon � Board-certified in Hematology and Medical Oncology (France)

� Professor of Medicine in Cell Biology. School of Medicine. Aix-Marseille University, Marseille, France

� Full Member. Institut Paoli-Calmettes, Comprehensive Cancer Centre at Marseille, France. Head: Cell Therapy Facility and Biological Resource Centre in Oncology.

� Member. Unité Mixte de Recherche (UMR) 1068 Inserm – CNRS – Aix-Marseille University – Institut Paoli-Calmettes. Centre de Recherches en Cancérologie de

� Marseille (CRCM)

� Scientific Coordinator. Centre d’Investigations Cliniques en Biothérapie (CBT-510) Inserm – Aix-Marseille University – Institut Paoli-Calmettes – Assistance Publique des Hôpitaux de Marseille

� Director. Cancéropôle PACA, PACA Regional Cancer Research Council

� Past-President of JACIE Executive Committee

� Chair EBMT Cell Processing Committee

� Member: AACR, ASH, EBMT, EHA, ESBB, ESMO, ISCT, SFGM-TC

42

Stefaan Van der Spiegel Stefaan Van der Spiegel coordinates the work of the European Commission's team on Substances of Human Origin (SoHO), including blood, organs and tissues&cells. Prior to that, he worked as consultant with McKinsey&Co for several actors in the healthcare sector and as policy officer on pricing and reimbursement of pharmaceuticals for the European Commission's Pharmaceutical Forum. He is trained in medicine (MD) and in business administration (MBA).

43

Marc Pittie Marc is partner at Bredin Prat, in charge of its Brussels office. Marc is registered at the Brussels and Paris bars. Marc is practicing EU law and, in particular, Competition Law. He is advising international clients in various sectors before the EU Commission and national competition authorities. Marc is also acting for his clients before national and European Courts in these fields. Marc holds a licence in Law, and a licence in Economics / University of Louvain. He speaks French, Dutch and English. Professional experience:

Since 03/2003: Partner at Bredin Prat - in charge of the Brussels office

11/1999 - 02/2003: Senior Associate at Allen & Overy / Brussels + Paris

04/1995 – 10/1999: Legal clerk for Mr Justice Koen Lenaerts at the Court of First Instance in Luxembourg (now the EU General Court)

09/1993 -03/1995: Associate at Loeff Claeys Verbeke

01/1993-04/1998: Assistant in the Consumer Law Centre and in the Contract and Tort Law department at the Law University of Louvain

44

Gilbert Verbeken Gilbert Verbeken (°1961) studied at Ghent University (Belgium). As a biologist, he is more than 25 years active in the field of human cell- and tissue (processing and) banking. Focusing on skin and keratinocytes. Since 1988, he is working at the Queen Astrid Military Hospital (Brussels, Belgium). Today, next to some operational activities related to the Hospitals’ Banks, he is QA/QC/RA Manager for these Banks (Skin, Keratinocytes, Hearth Valves, and Blood Vessels). As a Senior Scientist he is also involved in a (KHID) research project focusing on the development of biological skin equivalents. Gilbert has built up extra experience working together with biotech industry (shared projects) on “products” that are called Advanced Therapy Medicinal Products (ATMPs) today. He is appointed expert advisor to the Belgian Superior Health Council (Human Bodily Material), to DG Sanco (Biological Safety) and to DG Research (Ethics). He is affiliated (scientific co-operator) to the Free University of Brussels, Belgium (VUB) and to the University of Leuven, Belgium (KU Leuven). Gilbert also assisted the recent development of the European Good Tissue Practices Guidelines (Euro GTPs).

45

Scott A. Brubaker Since August of 2004, Scott has held the position of Chief Policy Officer at the American Association of Tissue Banks. His main duties are to liaise with the US FDA, the CDC, and other organizations nationally and internationally who are standards-setting or regulating bodies for cell & tissue banking. He also serves as the office liaison for the AATB’s Standards Committee and Physicians’ Council, and leads the Association’s membership in the creation of new standards and guidance documents. Oversight of the AATB Accreditation Program, including inspections, is a new responsibility. Prior to joining the AATB staff, he acquired 18 years of practical experience in tissue banking and organ donation at LifeNet in Virginia Beach, Virginia. Scott regularly provides educational presentations to promote best practice and tissue safety.

46

Esteve Trias i Adroher Transplant Services Foundation Tissue Bank – Hospital Clínic Barcelona

Education background

• Graduated in Medicine by University Barcelona 93

• Specialist in Emergency Medicine. University Barcelona 99

• Investigator Sufficiency University of Barcelona 96

• European Transplant Coordinator

• MBA-ESADE. Rovira I Virgili University 2004

Professional experience

• Medical Director, Transplant Services Foundation – Hospital Clínic Barcelona 2006- Tissue Bank.

• Technical Director of the Cell Therapy Factory – Hospital Clínic Barcelona – GMP certified by the Spanish Health Ministry

• Past-President of the European Association of Tissue Banks

• Expert from CEN- Center European Normalization, EC project European Codification of TC 2007-08.

• Expert and Advisor of the Portuguese Health Ministry of Health on Tissue Banking.

• Expert and Advisor of the Croatian Health Ministry of Health for the European Commission on Tissue Banking and Inspections.

• Member of Editorial Board Fundació Catalana de Transplantaments 2002-

• Member of the Medical Advisory Group of the EEBA – European Eye Bank Association

• Leader of the Standards Committee of the EATB – European Association of Tissue Banks through the Good Tissue Practices Project – GTPs

• Co-Chair of the EDQM group - Council of Europe for the “Guide for safety and quality for tissues and cells”.

• Member of the Technical Commission for Tissues. Spanish Health Ministry - ONT

• Member of different governmental commission’s Regulatory affairs on Tissue Banking, coding, Surveillance, Biovigilance, Safety and Quality matters. Catalan-Spanish Minister Health

Scientific activities

• Coordinator of Good Tissue Practices, SANCO project of EC 2008-11)

• Leader of Accreditation group “European Quality System on tissue Banking” SANCO project of the European Commission 2004-07

• Leader of Tissue Banking - EUROCET e-ten project EC2005-07

• Participate in more 15 different research projects.

• Participate in more than 50 invited lectures in different International and National meetings related with Tissue and Organ Donation and Tissue Banking.

• More than 30 International papers and 12 book chapters over organs-tissue transplantation.

Scientific Associations

• Past-President of European Association of Tissue Banks - EATB

• Member of the Board of Directors of the European Eye Bank Association - EEBA

• Member of the Board of Spanish Association of Tissue Banks - AEBT

• Member of the American Association of Tissue Banks - AATB

• Member of the European Transplant Coordinators Organization - ETCO

Education Activities

• Director, Coordinator, Tutor and/or Teacher of more than 50 different Training programs, national - internationally, face to face and on-line, different Universities and European Commission programs.

47

Ruth Warwick Ruth Warwick qualified in Medicine from Bristol University UK (1973) and specialized in clinical haematology (Royal Free, Hillingdon & Hammersmith Hospitals). She worked in Transfusion Medicine and as Consultant Specialist for Tissue Services for the National Health Service Blood & Transplant (NHSBT) from 1993 to 2010. She is a Fellow of the Royal College of Physicians MRCP 1977, FRCP 1997, & is a Fellow of the Royal College of Pathologists MRCPath 1980, FRCPath 1992. She is a past President of the British Association for Tissue Banking (2006-2008) and a past President of the European Association of Tissue Banks (2007-2009). She was Chairman of the European Commission CEN ISSS Workshop on Coding & Traceability of Tissues & Cells (2007-2008). She has had national & international experience in policy setting; was founder Chair of the National Institute for Biological Standards & Controls /UK Blood Services Tissues Advisory Committee for Tissues and Haemopoietic Stem Cells (1995-2001), was Consultant for the Council of Europe Expert Group on Organs, Tissues & Cells (1999- 2001) and also on its editing group for the first Guide for the Quality and Safety of Tissues and Cells 2012. She has had advisory roles with the English Department of Health Transplant Policy Unit & Human Tissue Authority. She has over 120 published papers, 6 edited volumes including lead editor of two multinational books in Tissue and Cell Donation and Usage (published 2009 and 2012), chapters, and letters as well as over 110 abstracts. She has been a member of the International Council for Commonality in Blood Banking Automation, ICCBBA Board of Directors since 2010 and its Chairman since 2012.

48

Ioana Siska

Dr. Ioana SISKA is a medical doctor, senior specialist in laboratory medicine, with a PhD in physiology. She worked as senior lecturer at the Victor Babes University of Medicine and Pharmacy in Timisoara -Romania until 2007, when she joined the European Commission. She worked as Scientific Officer in the Directorate General for Research and Development and from November 2010 she is the Policy Officer responsible for the EU legislation in the field of tissues and cells for human application within the Health and Consumers General Directorate.

49

Deirdre F. Fehily Deirdre is an inspector and technical advisor for tissues and cells at the Italian National Transplant Centre (CNT) and she represents CNT at the Regulatory Committee and Competent Authority meetings for Directive 2004/23/EC and on various Sanco expert committees. She was technical co-ordinator of the Italian-led European Union funded projects SOHO V&S (Vigilance and Surveillance of Substances of Human Origin) and EUSTITE (European Union Standards and Training for the Inspection of Tissue Establishments). She is a member of the Eurocet 128 team that was awarded the contract to develop the EU compendia of tissue establishments and tissue and cell products for Single European Coding System. Deirdre also provides technical support and advice to the World Health Organisation as a member of the WHO Transplantation Advisory Committee and is a member of the co-ordinating team for the Notify Project for Global Vigilance and Surveillance of Organs, Tissues and Cells (www.notifylibrary.org). She has participated as a course designer and tutor on numerous national and international training courses for EU tissue and cell inspectors and vigilance officers and for tissue and cell professionals. Prior to joining the CNT in Italy, Deirdre was Head of Tissue Services for the English National Blood Service. She has published numerous articles on tissue and cell banking and vigilance and recently was editor of two books published by Wiley Blackwell, Tissue and Cell Donation: An Essential Guide and Tissue and Cell Processing: An Essential Guide.

50

Marta López Fraga Marta López Fraga, Ph.D., is the Scientific Officer in charge of the organ, tissue and cell donation and transplantation activities at the European Directorate for the Quality of Medicines & Healthcare (EDQM)/ Council of Europe. She received her B.Sc. in Biology from the Complutense University of Madrid and a Ph.D. in Immunology from the Autónoma University of Madrid (Spain). She was a postdoctoral fellow at La Jolla Institute for Allergy and Immunology in San Diego (USA), working on the cellular regulation of T cell immunity and tolerance through co-stimulatory molecules. In 2006, she joined Neurome Inc. of the University of California, Riverside (USA), to work on the development of targeted mucosal vaccine delivery technologies. In 2008, she became a Senior Scientist with Sylentis; a Spanish biotech company that focusses on the development of new drugs based on RNAi technology. Since 2011, she has been in charge of the Council of Europe’s European Committee on Organ Transplantation (CD-P-TO); the Steering Committee responsible for transplantation activities at the EDQM. The CD-P-TO actively promotes the non-commercialisation of organ donation, the fight against organ trafficking and the development of ethical, quality and safety standards in the field of organ, tissue and cell transplantation. Its activities include the collection of international data and monitoring of practices in Europe related to the donation and transplantation of organs, tissues and cells with regards to quality, safety and ethical standards and their implementation. The CD-P-TO is also involved in the transfer of knowledge and expertise between organisations and experts through training and networking , the elaboration of reports, surveys and recommendations and the promotion of organ, tissue and cell donation for transplantation among the general public.

51

Martin Oliver Hildebrandt Office address: Technical University Munich, Faculty of Medicine TUMCells Interdisciplinary Center for Cellular Therapies

Trogerstrasse 9, D-81675 Munich, Germany Tel.: +49 89 41407810 Fax: +49 89 41407809 e-mail: martin.hildebrandt@lrz.tum.de Education and Professional Training

1972 - 1976 Primary School in Berlin 1976 - 1984 Secondary School, Gymnasium Steglitz zu Berlin 1985 - 1993 Medical School, Freie Universität (FU) Berlin 1987 - 1988 Dept of Neuropathology, FU Berlin 1988 - 1990 Dept of Molecular Biology and Biochemistry, FU Berlin 1989 Washington University School of Medicine, St. Louis 1993 - 1996 Resident/Intern, Robert-Rössle-Klinik am MDC Berlin, Dept of Haematology,

Oncology and Tumor Immunology March 1994 Doctor’s Thesis (summa cum laude) 1996 - 2002 Research Fellow, Charité, Berlin 2002 Board Certification, Internal Medicine 2002 - 2009 Qualified Person and Head of Quality Control, Stem Cell Facility, Robert-Rössle-

Klinik, HELIOS Klinikum Berlin-Buch 2003 Board Certification, Blood Transfusion Specialties 2003 Venia legendi, Internal Medicine, Humboldt University Berlin 2005 Head, Intermediate Care Unit (IMC/ BMT), HELIOS Klinikum Berlin 2008 Board Certification, Haematology and Oncology 2009 Professor for Stem Cell Transplantation (Tenure Track), Hannover Medical

School, IFB-Tx, GMPDU Since 2011 Executive Director and Qualified Person, TUMCells, Faculty of Medicine Technical University Munich

Since 2004 Presidency, Guardini Foundation Berlin Since 2005 Federal Ethics Committee, State of Berlin, Head of Subcommittee Since 2007 Working Group on blood-borne pathogens, German Federal Ministry of Health Since 2009 Laboratory Best Practices Committee, ISCT

52

Michael Doser Michael Doser got his Ph.D. in Biology at the University of Hohenheim. Since 1990 he is working at the Institute of Textile Technology and Process Engineering in Denkendorf, Germany, since 1998 as Head of the Department of Biomedical Engineering, where he develops with his group biomaterials for implants, regenerative medicine and medical devices with special interest on cell-material interactions. Since 2001 he is also Deputy Director of the Institute. Dr. Doser is lecturer at the University of Stuttgart and the University of Tuebingen and since 2011 also honorary professor of the Universtity of Stuttgart. He is member of several material and Tissue-Engineering – Societies and actually Vice President of the European Society for Biomaterials. He also active member of standard organisations (DIN, ISO, ASTM).

53

Alexander Benedikt Leichtle Born 21. Jan 1977 in Wertingen (D) 1983-1987 Pestalozzi elementary school Gersthofen (D) 1987-1996 Grammar school Gymnasium bei St. Stephan in Augsburg (D).

University entrance qualification 1996-2002 Studies of human medicine at the Ludwig-Maximilians-

University Munich (D). State examination. 01. Jan. 2003- 10. Dec. 2008 Advanced training in laboratory medicine at the Institute of

Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics at the University Hospital Leipzig (D) (Director: Prof. Dr. med. Joachim Thiery).

Medical specialist for laboratory medicine 23.08.2005 Thesis: “Dr. med.” in laboratory medicine 10. Dec. 2008-28. Feb. 2011 Medical specialist for laboratory medicine at the Institute of

Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics at the University Hospital Leipzig (D) (Director: Prof. Dr. med. Joachim Thiery).

Since 01. Mar. 2011 Consultant for laboratory medicine at the Center of Laboratory Medicine, University Institute of Clinical Chemistry at the Inselspital Bern (CH) (Bern University Hospital, Director: Prof. Dr. med. Georg Martin Fiedler, MBA).

Member of: The American Heart Association, German Society for Laboratory

Medicine (DGKL) with the workgroup for bioinformatics and extra-analytical quality assurance, German Society for Newborn Screening (DGNS), Swiss Society of Clinical Chemistry (SSCC).

54

Michael Harder Michael Harder is Managing Partner of the company corlife, Hanover, Germany. Corlife develops implants and medical devices for cardiovascular surgery. Corlife, founded 2006, is both a tissue establishment as well as a medical device company. From 2000 until 2006, Dr. Harder was managing director of a biotech company focused on peptide drugs. There he was inter alia involved in the financing of subsidiaries, as well as licensing of products to U.S. companies. Between 1995 and 2000 he was head of the development unit for target research within Merck’s R&D organization. Dr. Harder received his Ph.D. in biotechnology in 1992 and conducted postdoctoral training in antibiotic research from 1992 - 1994.

55

Isabelle Martinache Isabelle has been sharing her time for 5 years between the Agence de la biomedicine in Saint Denis (near Paris) and the multi tissue bank in Lille. Phd in Pharmacy, she began her career in 1998 at the University Hospital of Lille as a quality manager in a cell therapy for diabetes department, that was completed and remains consistent with the biomonitoring mission. She became responsible person for the multi-tissue bank in 2004, with the assistance of her dedicated coach, Dr Alain Vanderkelen. In 2008, she was given the challenge of setting the strategy for human tissue as a scientific expert to serve the Management Board at the French Agence de la biomédecine, In the field of tissues, on a national level, she completes her work in providing professionals and researchers with collective answers; guaranteeing equity of access, ethics and transparency of these activities; organising information and awareness campaigns, and evaluating and publishing tissues activities in an annual report, and being involved in training and promotion.

56

Christian Oliver Fölsch

Born 24th May 1963 in Hamburg Married one child 1982 College Examinations in Hamburg

1983-1989 Study of Medicine University Hamburg

5/1989 Graduating in Medicine University Hamburg

8/1996 Senior House Officer and Registrar Prof. Stürz Department Orthopaedic Surgery Gießen

2/1997 Head of bone Bank Orthopaedic Department

12/1999 Specialist for Orthopaedic Surgery

1/2002 Registrar Prof. K.Schwemmle General Surgery and Thoracic Surgery University Gießen

7/2002 Registrar Prof. Stürz Orthopaedic Surgery University Gießen

8/2002 Specialist for General Surgery

1/2003 Registrar Prof.L.Gotzen Trauma Surgery Orthopaedics University Marburg

3/2004 Specialisation Trauma Surgeon

3/2004 Senior Registrar Spine Surgery Dr.P.Moulin SPZ Nottwil Switzerland

9/2004 Senior Registrar PD.Dr.Naumann Orthopaedic Department Augsburg Hessing Hospital

8/2005 Consultant Trauma Surgery Hessing Hospital Augsburg

11/2007 Specialisation in Children Orthopaedics

4/2008 Consultant Spine Surgery Orthopaedic Department University Marburg

12/2010 Consultant Spine Surgery and Children Orthopaedics

8/2011 Overseas Fellow British Orthopaedic Association

57

Kelvin G. M. Brockbank OVERVIEW: My personal mission is “engineering and preservation of biological materials for restoration of patient health”. Central to this mission is the definition and design of conditions for long-term storage and distribution that will make it possible for cell therapy products, tissues, organs and tissue engineered constructs to be available World-Wide, regardless of environmental conditions. I developed the tissue and kidney preservation technologies for two publicly traded companies, CryoLife and Organ Recovery Systems.

EDUCATION: BA (1973) and MA (1976) in Zoology from Trinity College, Dublin, Ireland. PhD in Experimental Pathology (1980) from the Medical University of South Carolina, Charleston, SC, USA.

CURRENT POSITIONS: Founder, President & Chief Science Officer, Cell & Tissue Systems, Inc. Adjunct Professor, Regenerative Medicine and Cell Biology, Medical University of South Carolina. Faculty Affiliate, Institute for Bioengineering and Bioscience, Georgia Institute of Technology.

RECENT HONOURS: Chairman of the Science and Technology Advisory Committee, American Association of Tissue Banks (2013-2014). Recipient of the “George W. Hyatt Memorial Award” for superior service in the fields of tissue banking and human transplantation. (2009).

RECENT SELECTED PUBLICATIONS from >500 papers, chapters, patents and presentations 1. Brockbank, K.G.M. 1. Methods for Ice-free preservation of tissues. US Patent #8,440,390, 05/14/2013. 2. Campbell, L.H., Taylor, M.J., Brockbank, K.G.M. Development of Pancreas Storage Solutions: Initial screening of cytoprotective supplements for β -cell survival and metabolic status after hypothermic storage" Biopreservation and Biobanking. Published on line (02/06/2013). 3. Parajuli, N., Campbell, L.H., Marine, A., Brockbank, K.G.M., MacMillan-Crow, L.-A. MitoQ blunts mitochondrial and renal damage during cold preservation of porcine kidney. PLoS ONE 7 (11), 2012. 4. Votteler, M., Layland, SL, Lill, G., Brockbank, K.G.M., Horke, A., Schenke-Layland, K. RNA isolation from fetal and adult human tissues for transcriptional profiling. Biotechnology Journal, October, 2012. 5. Brockbank, K.G.M., Schenke-Layland, K., et al. Ice-free Cryopreservation of Heart Valve Allografts: Better Extracellular Matrix Preservation In Vivo and Preclinical Results. Cell and Tissue Banking, published on line (01/03/2012).

58

Jean-Luc Rummens Institute address: Jessa Ziekenhuis Campus Virga Jesse Stadsomvaart 11 B-3500 Hasselt BELGIUM phone: 011/30.97.40 011/30.97.53 fax : 011/30.97.50 email : jean-luc.rummens@jessazh.be Education

1974-1981 Medical education and training at the Catholic University of Leuven 1981-1986 Clinical Pathologist

� hematology and blood transfusion � clinical chemistry � microbiology and infectious diseases

Certificates

• Chemotherapy in haematology and oncology. Catholic University of Leuven. 1985-1986.

• Infectious diseases.Boerhaave Commissie.March 1988.

• Hospital Infection Control. Catholic University of Leuven. 1989-1990.

• Advances in Haematology. Royal Postgraduate Medical school, Hammersmith Hospital, London. 1991.

• Diagnostic Haematopathology. Royal Postgraduate Medical school, Hammersmith Hospital, London. 1992.

• Immunophenotyping in diagnostics. Erasmus Universiteit Rotterdam. May 1994.

• Quality control in medical laboratories (1994-1995) - U.I.A. (University Antwerp)

• Molecular pathology. Boerhaave Commissie – University of Leiden. 2000 Positions held

1980-1981 Internship in Dept. of Hematology at the Catholic University of Leuven 1981-1986 Resident Dept. Clinical Pathology at the Catholic University of Leuven 1986-today Clinical laboratory, Jessa ziekenhuis, Hasselt 1995-today Medical supervisor and director of the laboratory of stem cell processing,

collection & transplantation 1996-today University of professional education (KH¬Lim) of Hasselt Hematology, immunology and pathology 1999-today Director of the Clinical laboratory, Jessa ziekenhuis, Hasselt 1999-today Medical Supervisor of the laboratory of experimental hematology, Jessa ziekenhuis, Hasselt 2002-today Clinic monitor, tutor and promotor at the University of Hasselt, Diepenbeek –

Faculty of Medicine and School of Life Sciences, Hasselt University, Biomedical Research Institute and Transnational University Limburg

2003-today Clinical Director, Dept. of laboratory medicine, Jessa ziekenhuis Hasselt 2008-today Professor at the University of Hasselt, Diepenbeek – Faculty of Medicine and

School of Life Sciences, Hasselt University, Biomedical Research Institute and Transnational University Limburg

59

Marie-José Tassignon Is full professor of ophthalmology at Antwerp University, Faculty of Medicine and Chair of the Department of Ophthalmology at Antwerp University Hospital since 1991. She is member of 8 national societies and board member of 6. She is member of 18 international societies and board member of 6. She was invited speaker in many national and international societies, promotor of 6 PhD theses and published 215 articles in peer-reviewed journals. She is author of 6 patents, inventions related with cataract surgery and developed ophthalmic instruments in collaboration with the industry. She received several awards from which the Binkhorst Medal Lecture (ESCRS, Vienna, 2011).

60

Daniel Pipeleers

Daniel Pipeleers (1946 – B) holds an MD and PhD from Brussels Free University-VUB. Daniel was trained in diabetes research as investigator of the Belgian Fund Scientific Research and as Harkness Fellow of the Commonwealth Foundation (New York) at Université Libre de Bruxelles, Washington Univ-St Louis-USA and the Queen Elisabeth Foundation Brussels. He is full professor at VUB since 1980. Daniel is the founder and director of the Diabetes Research Center (DRC) and director of the JDRF Center for Beta Cell Therapy in Diabetes. He has authored over 200 publications in the field of beta cell biology, pathology and therapy in diabetes. Daniel Pipeleers is member of the Belgian Royal Academy of Medicine and Honorary Doctor at Uppsala University Sweden.

61

Stefaan Van Gool Stefaan Van Gool studied medicine, pediatric specialty and pediatric oncology subspecialty at KU Leuven. His PhD training at KU Leuven was in the field of cellular immunology. He received further training in pediatric neuro-oncology in Münster Germany. He is now clinical head for pediatric neuro-oncology, full professor at KU Leuven and guest professor at university of Saarland. He holds a mandate of senior clinical investigator at the Fund for Scientific Research – Flanders. He is the chair of the laboratory of pediatric immunology. He installed and leads the immunotherapy platform Leuven. In relation to his activities in the field of tumor immunology, he is KU Leuven holder of the Olivia Hendrickx Research Fund, the James E. Kearney Foundation, LCH Belgium and the Leuven’s ARCH fund.

62

Denis Dufrane

NAME

Dufrane Denis

POSITION TITLE

Head of Tissue/Cells Therapy Center, University clinical

hospital Saint-Luc, Brussels, Belgium

EDUCATION/TRAINING

INSTITUTION AND LOCATION DEGREE YEAR(s) FIELD OF STUDY

Catholic University of Louvain M.D. 2000 Medicine

Catholic University of Louvain M.Sc. 2001 Cellular and Molecular Biology

Catholic University of Louvain Ph.D. 2006 Cell Transplantation/ Encapsulation

Positions

2009-present: Medical director of the Endocrine Cell Therapy Unit (Islets and Adipose Stem

cells Bank) – University Clinical Hospital Saint-Luc; Catholic University of Louvain. 2009-present: Head of Bone Tissue Bank; University Clinical Hospital St-Luc, Catholic University

of Louvain. 2011-present: Head of Tissue/Cells Therapy Center, University Clinical Hospital St-Luc, Catholic

University of Louvain. Selected peer-reviewed publications.

(I) 40 published manuscripts

(II) 116 Presentations=

73 : Oral Presentations

43 : Poster Presentations

76: First author

01: Co-author

39: Last author

(III) 27 lectures sur invitation

63

Patrick Celis Patrick Celis joined the European Medicines Agency (EMA) in 1997. He holds a degree in Pharmacy and a PhD in Pharmaceutical Sciences from the University of Leuven, Belgium. Since December 2008, P Celis is responsible for the Scientific Secretariat of the Committee for Advanced Therapies (CAT). Before become Head of the CAT Secretariat, P Celis was responsible for the coordination of EU authorisation procedures for biological/biotech products and for the following scientific projects: Pandemic influenza vaccines, TSE, viral safety of recombinant products and Cell-based medicinal products. He held the positions of Scientific Secretary of the Vaccine Working Party and the Cell-based Products Working Party. Since 2006, he was involved in the implementation of the Regulation on Advanced Therapy Medicinal Products at the EMA. Prior to joining the EMA, P. Celis worked for 2 years as Pharmaceutical Assessor at the Belgian Ministry of Health.

64

Tyrone L. Pitt

BSc, MPhil, PhD Formerly Deputy Director of the Laboratory of HealthCare Associated Infections (retired 2009) in the Public Health Laboratory Service, subsequently the Health Protection Agency, England. Responsible for reference microbiological services providing assistance to hospitals for the identification and epidemiological tracing of opportunist bacteria from incidents and outbreaks of infection. Principal research interest was the pathobiology of Gram negative bacteria in chronic infections and the application of typing methods for the definition of bacterial populations. Published over 150 research papers and chapter contributions to books. Served on several editorial boards and presently Associate Editor of Epidemiology and Infection. Currently, part time Bacteriology Consultant to the National Health Service Blood and Transplant advising on the detection and prevention of bacterial contamination of blood and tissue products and investigation of suspected transfusion and transplant transmitted infections.

65

Dragoslav Domanovic MD, PhD, transfusion medicine specialist Senior Expert Vigilance and Traceability of Tissues and Cells of Human Origin European Centre for Disease Prevention and Control, Stockholm, Sweden I am medical doctor, specialist in transfusion medicine with a postgraduate academic degree in basic medical sciences. After couple of years working as a general practitioner, I continued my professional carrier as a specialist of transfusion medicine in the regional hospital and later in the National Blood Transfusion Centre of Slovenia in Ljubljana. There I was working in the field of blood cell separation and collection by apheresis and immuno-magnetic purging. At Medical faculty in Ljubljana I got a postgraduate academic doctoral degree for research in cells and tissues therapies and hematopoietic stem cell transplantation. I was actively involved in several national and international projects such as Optimal use of blood and Domaine and became a member of international associations for blood transfusion and cells and tissues therapy. As a Head of the Department for Blood supply and the public cord blood bank at the National Blood Transfusion Centre of Slovenia I have also been working on developing of the European and national legislation for blood, and tissues and cells. Currently, I am working at ECDC in Stockholm, Sweden as a senior expert for vigilance and traceability of tissues and cells of human origin with the main activities and responsibilities in scientific advice in transmission of communicable diseases through substances of human origin. This entails the developing of the risk assessments, opinions, positions and guidance as well as a close cooperation with relevant national Competent Authorities for tissues and cells, blood and organs. Additionally, I am exploiting the epidemiology of donor derived infections and the prevention and control of blood borne viruses especially through developing an European Framework for the prevention and control of hepatitis B and C.

66

Veroniek Saegeman Veroniek Saegeman is adjunct clinical head in microbiology at the Department of Laboratory Medicine and the Department of Hospital Epidemiology and Infection Control of University Hospitals Leuven, Belgium. She performed her PhD on the microbiological contamination and decontamination of human tissue allografts. Her focus subjects are infectious serology, diagnostics of mycobacterial infections, and screening for multidrug resistant microorganisms (MDRO). Research focuses on the detection of ways to prevent transmission of these MDROs. For instance, can point prevalence surveys in- and outside the hospital give an indication of the reservoir of these MDROs. Because of the emergence of highly resistant microorganisms and the lack of new antimicrobials, prevention is an important tool.

67

Fewzi Teskrat Fewzi TESKRAT is a Medical Doctor who works for the French Health Product and Safety Agency (ANSM) since 1996 (former Afssaps before 2011). ANSM is entrusted with guaranteeing the safety of healthcare products for human use throughout their entire life cycle (medicines, biological products, medical devices, devices for in vitro diagnosis, cosmetic products, tattooing products, biocide products, etc.). It assesses the safety of use, the efficacy and the quality of these products. It performs surveillance and laboratory testing of these products and conducts inspections of the manufacturing sites.

Dr Fewzi Teskrat is a senior inspector and technical advisor for human products and in charge of European and International affairs. He has developed a specific methodology for the inspection of procurement sites and tissue establishments, and also for organisations performing clinical trials. He has been involved in drafting the French legislation and guidelines in the field of tissue and cells for transplantation (good tissues and cells practices, decree, ministerial order, etc) and other regulations at European and international level. He represents the French Tissue and Cell Competent Authority (ANSM) at various expert and regulatory committee meetings and working groups. He provided technical support in the field of inspection of tissue establishments in particular during twinnings between ANSM and other Competent Authorities of EU Members States or third countries. He was an active member of several EU-funded projects in the field of blood and tissues and cells (i.e : EUBIS, CATIE, EUSTITE, SoHO & VS). Currently, he is the co-chairman of the PIC/S (Pharmaceutical Inspection Cooperation Scheme) expert circle on blood and tissues.

In the last past four years, he became more concerned about the growing threat of illegal and fraudulent activities in the field of organs, tissues and cells. Therefore he initiated and organised in collaboration with other national and international organisations (i.e. Interpol, Europol, European Commission, MS Competent Authorities) a seminar in April 2013 for raising awareness on illegal and fraudulent activities and finding solutions for their prevention. He has been invited by Interpol for its 81st general assembly to present his work on this matter and to establish a network between the Law Enforcement Officials (Customs and Police) and inspectors of Competent Authorities.

68

Wojciech J. Marczyński Head of Orthopaedic Division Postgraduate Medical Education Centre in Warsaw Teaching Hospital in Otwock Past President Polish Orthopedic and Trauma Society Date of birth: 18.09.1947r Number of publication – 168 I was the head of the Children Orthopaedic Department (1994 – 2001r), and followed by the head of the Trauma Department (2001 – 2007r). I promoted 16 PhD in medical science and one postdoctoral (now professor). I am the author and co-author of four books, the editor of the Polish edition 12 books, two patents and three utility models, I got a total of five awards research Rector of the Military Medical Academy. Award of the Minister of Science and Higher Education in 2012, Award of the Ministry of Health of Ukraine for training surgeons Ukraine 2010 y Award of the Ukrainian Society of Orthopaedics - Traumatology 2011 "for significant contribution to the development of the Ukrainian - Polish co-operation orthopaedic surgeons and traumatologists", member of the Academy of Sciences of Ukraine in 2011, Award of the Ministry of Health of Kazachstan for training surgeons Kazachstan 2011 y Member of the Scientific Council of the five scientific journals I use frozen bone grafts in clinical practice since 1981.

69

Thomas Rose

Personal data Male, °03 May 1965, Darmstadt, Germany

Education

1984: Secondary School: Gymnasium, Germany

1984-1986: Army Service as Officer, Germany

1986-1989: Nursery studies St. Elisabeth Hospital Darmstadt

1989-1991: Study of History, Geography and Chemistry at the

Technical University of Darmstadt, Germany

1991-1998: Study of Medicine, Free University Brussels

Practical Experiences and Training

1998/1999: Assistant Surgery, St. Nikolaus Hospital Eupen, Belgium

1999/2004: Assistant Surgery, AZ-VUB, Free University Brussels

2004/ : Certified General Surgeon

2004/2005: Intensivist in training, AZ-VUB

2005/2006: Residency Intensive Care Unit, AZ-VUB, Brussels

2005/2006: Clinical Tutor Intensive Care

2006/ : Senior Burn Surgeon, Senior Staff ICU, Adj. Medical

Director Skin- and Keratinocyte Bank, Military Hospital

Queen Astrid, Brussels

2007/ : Adj. Director of the Burn Unit, Medical Director Skin- and

Keratinocyte Bank, Military Hospital Queen Astrid, Brussels

2008/ : Member of the medical council Military Hospital Queen

Astrid, Brussels

2008/ : Certified MD in Intensive Care Medicine

2008/ : Certified Postgraduate in Nutrition

2008/ : Certified ABLS Provider Course, ABA Chicago

Membership Professional Associations

Royal Belgian Society for Surgery – RBSS

European Society of Intensive Care Medicine - ESICM

Vlaamse Vereniging voor Klinische Nutritie en Metabolisme – VVKNM European Tissue Repair Society – ETRS Board Member European Society of Parenteral and Enteral Nutrition – ESPEN American Society of Parenteral and Enteral Nutrition - ASPEN American Burn Association – ABA European Burn Association – EBA American Association of Tissue Banking – AATB Belgian Association of Burn Injuries - Executive board member as secretary Belgian Society of Intensive Care Medicine – SIZ International Society of Burn Injuries - IBSI

70

Diego Ponzin Diego Ponzin, MD is Director and Medical Director of the Veneto Eye Bank Foundation in Venice, Italy. He is Corneal Consultant in Venice as well. As Clinical and Surgical Advisor on the diagnosis and therapy of ocular surface diseases, his clinical fields of interest include the ocular surface and corneal diseases, cornea biology, storage and selection for transplantation. He obtained his medical degree at the University of Padua, Italy and thereafter, his post-doctoral diploma in Ophthalmology at the University of Udine, Italy. He has been Research Associate at the Department of Immunology of the FIDIA Research Laboratories in Padua. As fellow, he followed training at the Ophthalmic Division of Ospedale Umberto I in Venice-Mestre, Italy. Since 2004, he has been Expert for eye banking of the Consulta Nazionale Trapianti. During his career, he received several honours, including the Fidia Research Award (1991), the Fellowship “Franco Lenzoni” from the Associazione Italiana Donatori Organi (1996). He was Honorary Member of the Italian Society for Corneal Transplantation (2000) as well, and obtained the Scientific Research Award from the Italian Society of Ophthalmology in 2005 and the Medal of the Italian Society for Corneal Transplantation (2013). Dr. Ponzin is member of the American Academy of Ophthalmology and the Paton Society of the Eye Bank Association of America. He was member of the Medical Advisory Board and the International Relations Committee of the Eye Bank Association of America. He also was (vice) president of the Italian Society for Corneal Transplantation, the Italian Association of Eye Banks and the European Eye Bank Association. He participated as invited speaker in more than 200 Italian and international meetings on the subject of pharmacology, immunology, eye banking, and corneal and ocular surface diseases. Besides his involvement as reviewer of several journals, he is (co-)author of 50 full papers in peer-reviewed journals, 10 book chapters and 10 invited reviews. In the spare time: bass guitar player, writer (four books of novels and poetries, one Compact Disk, published).

71

Jan Lerut Cliniques Universitaires St-Luc, Brussels Université catholique de Louvain (UCL) Department of abdominal surgery and transplantation Brussels, BELGIUM Jan LERUT, MD, PhD (February 18, 1951) trained in General Surgery at the Katholieke Universiteit Leuven (KUL) (B), the H.Heine University of Dusseldorf (G) and at the Université catholique de Louvain (UCL) (B). From the very start of his surgical career he was involved in organ transplantation. This interest resulted in a transplantation fellowship at the Universities Paris-Sud - Centre Hépatobiliaire under the lead of Prof. Bismuth and Pittsburgh Medical Centre under the lead of Prof. Starzl. He was director of the abdominal transplant program at the Inselspital University of Bern (CH) from 1987 to 1991. Currently he is ordinary Professor of Surgery, Director of the Starzl Abdominal Transplant Unit of the University Hospitals Saint Luc of the Université catholique Louvain (UCL) and Director of the UCL Transplant Center in Brussels. He is also director of the department of abdominal and transplantation surgery at the same university. He has served as president of the Belgian Society of Transplantation (BST), as chairman of the Eurotransplant (ET) Liver Allocation Committee (ELIAC) and as President of the European Society for Organ Transplantation (ESOT). He is member of different councils and learning societies related to surgery and transplantation. He is active in the Euroliver awareness campaigns for adolescents in relation to organ donation. He will organize with the BST the ESOT 2015 congress. Under his Presidency of the ELIAC, the MELD system was introduced within the ET Community. His presidency of ESOT was devoted to the broadening of the European transplant community and to the development of a master educational program in the field of transplantation. He has published over 220 peer-reviewed articles, authored 24 books chapters and 24 scientific films. He made more than 550 communications on national and international congresses, most of them devoted to liver transplantation. His research interests focus on the development of technical refinements in liver transplantation and on the use of minimal immunosuppression and tolerance induction in liver transplantation.

72

Izabella Anna Uhrynowska-Tyszkiewicz Warszawski Uniwersytet Medyczny / Medical University of Warsaw, Poland Krajowe Centrum Bankowania Tkanek i Komorek / National Centre for Tissue and Cell Banking, Warsaw, Poland Born in Lodz, Poland. Graduated from the Medical University of Warsaw (MUW) with M.D. in 1999. From the very beginning her scientific career has been related to activities of the Department of Transplantology and Central Tissue Bank of the MUW. Pursued a Ph.D degree in 2008 at the MUW. Holds a position as Assistant Professor at the MUW. Author and co-author of several dozen of publications (conference abstracts, journal articles and book chapters). Engaged in creation and development of the Krajowe Centrum Bankowania Tkanek i Komorek (KCBTiK, National Centre for Tissue and Cell Banking) - the Competent Authority for tissues and cells in Poland. Holds a position as Deputy Director for Medical Affairs at the KCBTiK.

73

Hovan Chenorhokian Born 09 January 1983 in Paris, France 2008: graduation from medical school (Lariboisière, Paris, France) From 2008 to 2013: Surgical residency in general and vascular surgery (Lille, France) From 2013: Medical degree, Fellowship in vascular surgery (Lille, France)

74

Gebrine El Khoury Professor of Cardiac Surgery. Head, Department of Cardiovascular and Thoracic Surgery, Cliniques Universitaires Saint-Luc UCL, Avenue Hippocrate 10/6107, 1200 Brussels (Belgium). Medical education followed at the Catholic University of Louvain (Université Catholique de Louvain) - 1200 Brussels (Belgium), 1977-1984, cum Maxima Laude. He was First Laureate of the Special Prize (Medical School of the Catholic University of Louvain). E.C.F.M.G. July 1986 Training in Cardiac Surgery followed at the UCL Saint Luc Brussels:

• 1984-1990: Resident

• 1990 - 1995: Department of Cardiovascular and Thoracic Surgery - Catholic University of Louvain - Cliniques Universitaires Saint-Luc (Brussels - Belgium)

In 1995 he did a Clinical Fellow at the "Toronto General Hospital" Toronto (Canada) under supervision of Prof. T. David, where he was trained on the aortic valve repair. During the same year he did an internship at the Department of Cardiac Surgery "Hôpital Broussais" Paris (France), where he was trained by famous Prof. A. Carpentier. In September 1996 he was appointed the Associated Professor at the Department of Cardiovascular and Thoracic Surgery - Catholic University of Louvain - Cliniques Universitaires Saint-Luc (Brussels - Belgium). He is world-wide recognized specialist for valve repair surgery, particularly in cases of bicuspid aortic valves. He has been Chair of the International Congress “Aortic Valve Reconstructive Surgery” since 2000. He has a large experience in clinical application of the human cardio-vascular tissues (valves/arteries) and particularly in “ROSS operation” (20 years). Prof El Khoury has large series of aortic valve replacement with allografts in extremely complicated case with abscess formation and septic state. Also he has done a series of Mitral allografts for endocarditis of mitral and tricuspid valve.

75

Alain Prat

Né le 13 Octobre 1947 à Saint Mandé (Val de Marne) Adresse professionelle

Service de Chirurgie Cardiaque Hôpital Cardiologique CHRU 59037 LILLE CEDEX Tel : 03 20 44 53 56 Fax : 03 20 44 56 61 e-mail : alain.prat@chru-lille.fr

Fonctions actuelles

• Professeur des Universités- Praticien Hospitalier

• Chef du Pôle Cardio-Vasculaire et Pulmonaire

• Chef du Service de Chirurgie Cardiaque Hôpital Cardiologique – CHRU de Lille Université de Lille 2

Titres et fonctions universitaires

• Docteur en Médecine (1980)

• Chef de Clinique à la Faculté de Lille (1980 – 1984)

• Professeur des Universités (1998)

• Membre du Conseil National Supérieur des Universités (depuis 2004)

• Président de la sous section 5103

• 1er Vice Président de la 51ème section Titres et fonctions hospitaliers

• Interne des Hôpitaux de Lille (1974)

• Assistant des Hôpitaux de Lille (1980-1984)

• Médecin Adjoint (1984)

• Praticien Hospitalier (1985-1998) Fonctions représentatives

• Membre de la CME du CHU de Lille (1990 – 1994)

• Coordonnateur du Pôle de CCV du CHU de Lille (2001 – 2005; 2007 – 2010)

• Membre du Conseil d’Administration de la SFCTCV (depuis 2004)

• Chef du Pôle Cœur – Vaisseaux – Poumons (depuis 2010) Principales Sociétés Savantes

• Société Française de Chirurgie Thoracique et Cardio-Vasculaire (SFCTCV)

• Collège Français de Chirurgie Thoracique et Cardiovasculaire

• European Association for Cardio-Thoracic Surgery (EACTS)

• European Board of Thoracic and Cardiovascular Surgeons (EBTCVS)

76

Bart Meyns Address at work: Department of Cardiac Surgery Gasthuisberg University Hospital (KULeuven) Herestraat 49 3000 Leuven, Belgium Tel: 16 344260 Fax: 16 344616 e-mail: Bart.Meyns@uz.kuleuven.ac.be 1980 - 1987 Medical school at the Catholic University of Leuven (KUL) 31-7-1987 Doctor in Medicine (Genees- Heel- en Verloskunde) summa cum lauda 1987 - 1991 Residency in General Surgery in - Duffel, Belgium - Exeter, UK - Leuven, Belgium 1991 - 1993 Residency in Cardiac Surgery in Leuven, Belgium 31-7-1993 Recognition as General Surgeon, Belgium Recognition as Cardiac Surgeon, Belgium 22-5-1997 Doctor in Medical Sciences (PhD) on the scription ‘Ventricular support with miniature rotary blood pumps’ 1-10-1997 Appointed as Professor in Cardiac Surgery at the Catholic University Leuven.

Active in adult and pediatric cardiac surgery Research in the field of

mechanical support systems chronic heart failure congenital heart disease

2000 - 2002 Academic degree: ‘Hospital and Health management’. 31-07-2002 Recognition in ‘Hospital and Health management’ on the scription:

‘International cooperation in pediatric cardiac surgery’. 1-10-2007 Chairman of the Department Cardiac Surgery UZLeuven 1-12-2009 Chairman of the Department of Cardiac and Vascular Surgery UZLeuven 1-4-2013 Chairman of the ‘Care Program’ committee and member of the board of

directors UZLeuven. Published >150 papers in reviewed journals. Member of Royal Belgian Society of Surgery Belgian Society for Cardiothoracic Surgery (Board member - Former Secretary-General) European Association of Cardio-Thoracic surgery International Society for Heart and Lung Transplantation Belgian Society for Transplantation Association for European Paediatric Cardiology International Society for Rotary Blood Pumps (Board member - Past - President) European Society of Artificial Organs (Board member - Past - President) European Society for Congenital Heart Surgery

77

Samir Sarikouch Samir Sarikouch MD, PhD, Department for Cardiac-, Thoracic-, Transplantation and Vascular Surgery, Hannover Medical School, will act as Clinical Trial Director for ESPOIR and manage Work Package 4 -Clinical Trial and Regulatory Affairs. Dr. Sarikouch avails of significant experience in the management and implementation of clinical trials over many years, including three prominent clinical studies on Tetralogy of Fallot within the multidisciplinary network of the German Competence Network for Congenital Heart Defects.

78

Abstracts of oral presentations (Listed in order of timing of presentation)

79

O-001 – Truth and Transparency in Tissue Donation S. Bartlet

International Consortium of Investigative Journalists

Science and medicine has brought amazing advancements to the repair of bodies debilitated by aging, injury and disease with bone and tissue transplantation. However the ethical and regulatory structure has not kept pace. When problems arise and mistakes are made the damage to public confidence, and future donations is significant. An industry that relies on altruism has a responsibility to ensure that everyone involved in the donation process behaves with the highest ethical standards of honesty, transparency and respect. Hiding, ignoring or denying problems will not fix them. Journalist Sandra Bartlett will talk about the key findings of an examination of the human tissue industry by the International Consortium of Investigative Journalists. Skin And Bone: The Shadowy Trade In Human Body Parts is an eight-month project across 11 countries. The investigation relied on more than 200 interviews with industry insiders, government officials, surgeons, lawyers, ethicists and convicted felons, as well as thousands of court documents, regulatory reports, criminal investigation findings, corporate records and internal company memos.

80

O-002 – Self-sufficiency and the non-commercial nature of tissues of human origin L. Noël

World Health Organisation

Many arguments are used to defend the principle that the human body and its parts as such should not be the source of financial gain. They range from a conception of humanity where the body and its parts as such deserve such a respect that they cannot have a price, to the rejection of the loss of social coherence resulting from the use of money to attract vendors. Ultimately many put forward their emotional “repugnance” to make financial profit from a component of the human body. Yet the shortage in human cell, tissue or organ for clinical application leads to consider all possible approaches to meet patients’ needs, including financial incentives to obtain human body component for the preparation of medical products of human origin. Financial gains on their availability inevitably follows if it does not precede, at least where the regulatory oversight is weak. Can the use of money be disconnected from the emergence of markets, profits and profiteering? The non-commercial nature of the human body and its parts as such has the capacity to effectively meet patients ’needs provided unprecedented efforts are directed at associating the public and professional in what needs to be understood as a collective resource. Indeed societal values such as solidarity and reciprocity must be the driving forces of the procurement of components of the human body for clinical application if all patients needs must be equitably met. The self-sufficiency paradigm namely consist in meeting the needs of patients from a given population with an adequate provision of transplantation services and supply of organs from that population. With government support and oversight, the paradigm underwrites: (i) equity in donation from possible donors and equity in allocation; (ii) education about donation but also about prevention of diseases that create a need for transplantation; and (iii) transparency and professionalism.

81

O-003 – Collaborating with medical examiners and coroners to increase donation M. Haun,1 C. Parsons,2 J. Mohr,3 K. Lotherington3 1 Canadian Blood Services, Ottawa, Canada 2 Canadian Blood Services, Edmonton, Canada

3 Canadian Blood Services, Halifax, Canada

Introduction: Approximately 50% of deaths in Canada occur outside the hospital environment with most falling under medical examiner or coroner jurisdiction. In 80% of Canada’s tissue banks, less than 5% of donors are identified from death investigation cases, identifying a significant opportunity for improvement. Goal: In August 2008, the federal, provincial and territorial (F/P/T) governments gave Canadian Blood Services a mandate to work with the tissue donation and transplantation communities to develop leading practices and recommendations in support of system performance improvement. Methods: One national partner organization engaged early on by Canadian Blood Services was the Canadian Conference of Chief Coroners and Chief Medical Examiners, which led to the development of a Reference Manual for Donation for Canadian coroners and medical examiners as well as an initiative to implement practical steps to increase tissue donations from deaths outside the hospital environment. Canadian Blood Services is facilitating collaborative workshops with medical examiners and coroners in the Provinces of Manitoba, Ontario, New Brunswick, Saskatchewan and Nova Scotia, and in each province tissue recovery and organ procurement organizations, pathologists, funeral professionals and governments officials are joining these process improvement workshops, with a goal of increasing the identification and referral of donors. A panel of Canadian and international tissue banking and death investigation experts has also been convened to support this initiative. The first provincial workshops are taking place in 2013, the others will follow in 2014. Result: To-date, participants in the provincial workshops have identified several local opportunities for improvement and are improving or creating new functional linkages between organizations to realize more tissue donations. Knowledge gained through these provincial collaborations will be detailed in a report and shared with the broader Canadian donation community. Conclusion: Medical examiners and coroners can be engaged as key partners to support increasing tissue donation from deaths that occur outside the hospital.

82

O-004 – Cornea donation and allocation in the Netherlands: the Dutch perspective H. A. van Leiden, M. B. A. Heemskerk, S. P. J. van Brummelen Dutch Transplant Foundation, Leiden, The Netherlands

Introduction: In the Netherlands 708 patients (Sept. 2013) are on a national cornea waitinglist. The Dutch Transplant Foundation is assigned by law to register all cornea donors reported by medical doctors in the Netherlands, to check medical suitability, to organize procurement, and to allocate corneas to Dutch recipients. One of our objectives is equal allocation. Goal: Insight in the current waiting list and cornea flow from donation until allocation is needed. Methods: Data about cornea donation, waiting list and allocation stratified by type of cornea and transplant center, during the years 2010-2012 were evaluated. A waiting list model was created to simulate several alternative strategies of allocation. Results: The number of cornea donors increased from 1358 in 2010 to 1635 in 2012, due to an increase of the donor acceptance age limit from 75 to 85 years in October 2010. This resulted in an increase of corneas allocated to Dutch transplant centers from 1165 (57 HLA-typed PKP, 474 untyped PKP and 634 untyped lamellar) in 2010 to 1385 (52 HLA-typed PKP, 453 untyped PKP and 880 lamellar) in 2012. The national waitlist did not decrease concordantly due to increasing demand of untyped lamellair cornea. Waiting times for untyped cornea differed considerably between centers. Allocation of untyped corneas was complicated by different cornea bank preferences, planning of operation rooms, and waiting list management. In our model a 5% increase of cornea donations could in a few years halve the waiting time for untyped corneas, and the number of cancelled operations. No preference of cornea bank and flexible planning of operation rooms by transplant centers significantly decreased the number of expired corneas and flexible planning also decreased the number of cancelled operations. Conclusion: Waitlist modeling showed that the mean waiting time can considerably decrease with 5% increase of donations. Then, a more flexible operation planning for corneas already in stock, from both cornea banks, must minimize the number of expired corneas. Adapting the allocation management of corneas in the Netherlands could realize a better distribution of corneas with more equal waiting times.

83

O-005 – Cardiovascular tissue – extending donor age limits, UK experience J. Davies,1 M. Johnson2 1 Oxford Heart Valve Bank, Oxford University Hospitals, Oxford, UK

2 Heart Valve Bank, Royal Brompton Hospital, London, UK

Intro & Goal: UK Blood Transfusion guidelines1 for cardiovascular tissue donors is 60 years (aortic valves) and 65 years (pulmonary valves). Two non-blood service heart valve banks (Royal Brompton in London and Oxford University Hospital) process tissue from older donors to alleviate national tissue shortages. Review of this experience will be used to support proposal to extend UK donor age limits. Method: Brompton and Oxford retrieval data (2009 – 2013) was collated. National tissue donor data (2012) was used to predict additional supply. Tissue availability in UK (2013) reviewed to assess demand. Clinical application of tissue supplied 2011 - Aug 2013 was examined using recipient information and evaluated separately for cohorts of valve tissue, conduits and patches. Adverse incidents and surgical opinion was assessed using customer satisfaction surveys. Results: Brompton and Oxford currently process additional 8.8 aortic and 22.3 pulmonary grafts from donors 60 – 65 years. UK stock lists confirm 3 large aortics (>22 mm internal diameter) and 12 bifurcated pulmonary valves (> 10 mm internal diameter) available in August 2013. Older donors could provide an additional 25.9 aortic and 65.4 pulmonary grafts. Between Jan 2011 – Aug 2013 Oxford retrieved 3 aortic valve/conduits, 17 pulmonary valves, 25 pulmonary conduits and 16 pulmonary patches from donors of age 61 - 71 years. Oxford confirm 59 (96.7%) have been used. Recipient information for 53 (90%) tissues was reviewed. Average age of the recipients decreases; aortic graft (53 yrs), pulmonary valve (11.8 yrs), conduits (3.5 months), pulmonary patches (10 weeks). All aortic tissue was supplied to adult patients. Pulmonary tissue utilised as bifurcated patch (from conduits) or a square or rectangular patch. No adverse incidents reported. Surgeons comments include “I fully support the extension of the age limit as the alternative of Bovine or Porcine pericardium pickled in glutaraldehyde is far inferior” “We have had to recently start using decellularised Equine pericardium which is very pliable but utterly untested in a human model over time, using older human tissue is far superior”. Conclusions: Increasing UK donor age limits could provide significant amount of additional tissue in demand. Tissue quality from older donors is acceptable. Patient outcome studies could examine pulmonary tissue used as patches separately. This data endorses proposal to extend donor age limits similar to Europe2 and US3. References:

1. www.transfusionguidelines.org.uk/index.asp?Publication=CTD 2. Cell & Tissue Banking 2008.9: 31-36. Are heart valves from donors over 65 years morphologically suitable for transplantation? Grosse K, Meyer R, Schmitzer E et al. 3. www.aatb.org/AATB-Standards-for-Tissue-Banking

84

O-006 – Validation of Oxford Quality Assessment Procedure to extend cardiovascular tissue donor age limits J. Davies,1 S. Bertazzo,2 M. Johnson3 1 Oxford Heart Valve Bank, Oxford University Hospitals, Oxford, UK 2 Dept of Materials, Imperial College London, London, UK

3 Heart Valve Bank, Royal Brompton Hospital, London, UK

Intro & Goal: Two non-blood service UK heart valve banks (Royal Brompton in London and Oxford University Hospital) wish to extend cardiovascular tissue donor age limits1 to alleviate national shortage of tissue. Atherosclerosis containing calcium deposits are reported in more than 65% of the population over 40 years of age2. Aortic valves from older donors are likely to be discarded for morphological reasons. In order to ensure that only good quality grafts from older donors are released for clinical application, Oxford commissioned a study to validate their protocol for tissue quality assessment (QA). Method: Cardiovascular tissue was prepared for qualitative assessment by nano-analytical imaging using a proven scanning electron microscopy (SEM) method3, 4. Samples were prepared for SEM and imaging performed using SEM (Gemini 1525 FEGSEM) at 10kV with an inlens detector. Presence of calcium and atheroma was then quantitatively assessed by a novel digestion technique involving collagenase and Ripa buffer. Tissue was centrifuged, resulting pellet weighed and prepared for SEM. Results: Samples of 52 aortic, 5 pulmonary grafts were prepared for SEM imaging. Age range of donors was 0 to 81 years (mean 52.7 years), 43% grafts from female donors. Digestion technique was used to analyse 43 aortic grafts, donor age range 0 to 78 years (mean 42.5 years), 51% females. Density dependant colour SEM images confirmed presence of spherical calcium phosphate particles in rejected aortic tissue of older donors. Spheres were detected in young donors (rejected by QA) and absent in the great majority of aortic grafts from older donors (accepted by Oxford). Pulmonary grafts from older donors had the same amount of spheres as child donors. Aortic samples rejected by QA were examined by quantitative digestion technique, and had higher % tissue dry mass (>3 %). Risk factors for cardiovascular disease in the few donors with unusual results were reviewed. Conclusions: Quality assessment schemes may be endorsed by SEM and Digestion techniques. The older Oxford donors found to have absence of calcific aortic valve disease (CAVD) confirm that, although CAVD is more common with age, it is not inevitable. This study affirms extension of donor age limits5. If this Digestion technique is standardised, it could be used routinely to quantitatively assess atheroma and calcium accumulation. References:

1. www.transfusionguidelines.org.uk/index.asp?Publication=CTD 2. Calcific aortic valve disease: not simply a degenerative process. Rajamannan NM, Evans FJ, Aikawa E, Grande-Allen KJ, Demer LL, Heistad DD, Simmons CA, Masters KS, Mathieu P, O’Brien KD, Schoen FJ, Towler DA, Yoganathan AP and Otto CM. Circulation 2011, 124:1783-1791. 3. Nature Materials. 12, 576–583. (2013). Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification. Sergio Bertazzo S, Gentleman E, Cloyd K, Chester AH, Yacoub MH & Stevens MM. 4. Materials Research Society. Published online: 13 June 2013. Calcified Spheres: The Beginning of Cardiovascular Disease? by Michael J. Cohen. 5. Cell & Tissue Banking 2008.9: 31-36. Are heart valves from donors over 65 years morphologically suitable for transplantation? Grosse K, Meyer R, Schmitzer E et al.

85

O-007 – Requirements for a clinical a clinical grade cellular cancer vaccine – what it takes to get through M. Eyrich Cell Processing Facility, University Children’s Hospital Würzburg, University of Würzburg, Germany

Immunotherapy of patients with high-grade glioma using autologous, tumor-lysate pulsed dendritic cells (DCs) represents an important consolidation therapy in order to maintain remission after conventional therapy. Several phase I, single-center studies with a total of more than 500 treated patients so far have shown a favorable risk-benefit assessment and clinical efficacy in some patients. The conduct of larger, randomized studies requires the recruitment of more vaccination centers which is hampered by current regulatory restrictions. According to current EU-guidelines, DCs cultured over a period of 9 days are considered as advanced therapy medicinal products (ATMPs), tumor-lysate is classified as a tissue. Thus, orchestrating GMP-, GTP- and finally GCP-requirements represents a major challenge for academic institutions, which are interested in further development and translational research. Our institution is now working for more than 7 years to implement this innovative approach in multimodal treatment strategies for brain tumors. The current legal framework for such therapies in Germany, the challenges for academic institutions arising thereby and how these hurdles might be overcome will be the focus of the presentation.

86

O-008 – Consequences of the ATMP regulation in the field of Hematopoietic Stem Cell Transplantation C. Chabannon

Centre de Thérapie Cellulaire, Département de Biologie du Cancer, Institut Paoli-Calmettes & Inserm CBT-510, Marseille, France

“Regulation (EC) No 1294/2007 of the European Parliament and of the Council off17 november on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004 » creates a new class of medicinal products that include somatic cell therapy products, products derived from tissue engineering, and gene therapy products, that can be combined with biomaterials. As for somatic cell therapy products, they share similarities with cell therapy products that are prepared and delivered by cell therapy facilities and tissue banks regulated by national competent authorities; in particular somatic cell therapy products (ATMPs) and cell therapy products are prepared from the same starting material obtained from donors or patients. The field of Hematopoietic Stem Cell Transplantation (HSC T) has been very active over the last 50 years; significant changes have taken place in terms of indications, conditioning regimen, supportive care, donor selection, and cell procurement; while most autologous and allogeneic HSCT use minimally-manipulated cell products, tools are progressively becoming available to manufacture better defined hematopoietic cell products, and allogeneic cell transplantation is increasingly seen as a multi-step procedure in which the recipient receives various types of cell therapies in order to establish hematopoietic chimerism, then enhance immune anti-tumour and anti-infectious activities; more than minimally-manipulated – or substantially-manipulated – hematopoietic cell products however fall under the ATMP regulation, and can no longer be produced and manufactured by cell therapy facilities. The presentation will review current and future consequences of the implementation of the ATMP regulation on the delivery of HSCT, on the conduct of clinical research in this field, and on the organization of essential activities including clinical, collection and processing.

87

O-009 – The EU regulatory framework for quality and safety of human tissues and cells S. Van der Spiegel

European Commission

The EU legislation aims to establish common standards for quality and safety in all 28 EU Member States for substances of human origin, i.e., organs, tissues&cells and blood. The tissues&cells sector is a heterogenous sector, covering replacement tissues, reproductive medicine and haematopoetic stem cell transplants. While some parts of legislation are common for these 3 tissues/cells fields, they also require some dedicated and specific requirements. As for blood and for organs, the tissue&cells sector is regulated through dedicated Directives laying down a quality and safety framework with requirements on 3 levels: for healthcare professionals, for national authorities and for the European Commission. - Professionals are obliged to follow a set of quality measures and requirements in articular

on how to select donors, test for infectious diseases and process the donated substances. - National authorities need to establish thorough oversight of procurement activities and

establishments, including inspections, authorisations and the set-up of functional traceability and vigilance tools.

- It is the Commission's task to ensure that the legislation is correctly applied and provide a platform to enable interconnectivity between the Member States's traceability and vigilance tools.

The heterogeneity of the sector, together with scientific development (e.g. more bedside technologies or engineered tissues), increasing commercialisation of some substances (e.g. bone) and an increasing global flow of substances create new challenges for safety and quality that need to be addressed with adequate legislation. It falls therefore on the Commission to regularly assess the efficacy and implementation of the legislation, and where needed propose amendments. While still analysing results of an implementation survey answered by the 28 EU Member States, the Commission is already planning implementing legislation on a single coding system and on procedures to verify the quality and safety of tissues and cells imported from third countries.

88

O-010 – The concept of « Placing on the market » under the EU regulatory framework: some flexibility for ATMPs? M. Pittie

Bredin Prat Law Firm; Brussels, Belgium – Paris, France

Article 2 of the Directive 2001/83/CE related to medicinal products for human use and to the marketing authorization legislation stipulates that “the provisions […] shall apply to industrially produced medicinal products for human use intended to be placed on the market in Member States”. The analysis of the legislation and the case law revealed that these two conditions (“industrially produced” and “placed on the market”) might not be easily met for human cells and tissues such as the keratinocytes produced by the Queen Elizabeth Military Hospital in Brussels. They should therefore not be subject to the marketing authorization legislation. Moreover, if one were to consider that these human cells and tissues are industrially produced and that they are placed on the market, the article 5 of the Directive which enables Member States to exclude from the provisions of the Directive medicinal products supplied in response to a bona fide unsolicited order, formulated in accordance with the specifications of an authorized health care professional and for use by his individual patients on his direct personal responsibility should apply. In other words, we are of the view that the European Regulatory Framework offers some flexibility to further allow the non profit organizations such as the Queen Elizabeth Military Hospital to remain active in transplanting human cells and tissues for the main benefit of the patients and the EU Member States health care systems.

89

O-011 – EU-wide economic overview of the markets of tissues and cells for transplantation T. de By,1 I. Geesink,2 A. Bokhorst,3 M. Heerings,2 J. Stein4 1 Foundation of European Tissue Banks, Berlin, Germany, 2 Rathenau Institute, The Hague, The Netherlands,

3 Trip, The Hague, The Netherlands,

4 German Heart Institute, Berlin, Germany

Rationale: Due to a shortage of information, there is an insufficient understanding of the markets for human tissue and cells within the EU, as well as to/from the EU to third countries, to provide an insight in the traffic of human tissue. The lack of oversight hinders the European Commission to develop adequate policies and decision making to ensure a balance in demand and supply of voluntary and unpaid donations of safe tissues and cells for transplantation throughout European Union. Therefore, the EAHC has called for a study aimed at producing and investigation of the specific features and types of markets in the three medical sectors as outlined by the Commission: 1. Replacement tissues, 2. Stem cells from (cord) blood and bone marrow and 3. Gametes and tissues for assisted reproductive technology (ART). The study will address markets and key players in the field of transplantation medicine across the three respective domains: at a European level, and to some extent in relation to the US and relevant third countries. The study will focus on identifying current and emerging economic practices, key players in both public- and private sectors, legislative and reimbursement schemes across Member States, and finally on providing a forecast of both technological trends and of associated ethical, legal, and social issues. During the course of this study, the team will be in contact with some 80 to 100 stakeholders, either via interviews, fieldwork, surveys, the advisory panel or stakeholder meetings for discussion and presentation of results. For additional quality review and to secure access to difficult to reach sources and data, an advisory panel of high-level experts has been set up to support the project team. The outcome of the study will support the Commission in taking the next steps in providing adequate regulatory framework for the EU wide tissue & cells markets. Based on the present expertise of the consortium it is known that tackling dilemmas concerning adequate supply of tissues and cells within the EU, and the existence of multi- facetted cross border traffic of grafts and patients, is complex and challenging. Moreover, where shortage of human bodily material plays a role, an increase of commercialization may be observed. Whereas such shortage doesn’t seem to exist in some EU- and a number of third countries, an unbalanced economical relation may lead to an undesirable disadvantage for those patients, hospitals, and health care professionals who lack the means to obtain the grafts which are offered through these “commercial” channels.

90

O-012 – Queen Astrid Military Hospital’s 26-Year Old Human Keratinocyte Cultures Became Advanced Therapy Medicinal Products at the Dawn of the Third Millennium: Historical Reflection & Practical Impact of this Evolution

G. Verbeken,1 A. Vanderkelen,1 T. Rose,1 S. Jennes,2 J.-P. Draye,1 D. De Vos,1 J.-P. Pirnay1 1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology, Burn Wound Centre,

Queen Astrid Military Hospital, Brussels, Belgium 2 Burn Wound Centre, Queen Astrid Military Hospital, Brussels, Belgium REGULATION (EC) No 1394/2007 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 13 November 2007 on advanced therapy medicinal products (ATMPs) is a lex specialis, which introduces additional provisions to those laid down in Medicinal Products Directive 2001/83/EC. The scope of ATMP Regulation No 1394/2007 is to regulate advanced therapy medicinal products which are intended to be placed on the market in Member States and are either prepared industrially or manufactured by a method involving an industrial process, in accordance with the general scope of the Community pharmaceutical legislation laid down in Title II of Directive 2001/83/EC. ATMPs which are prepared on a non-routine basis according to specific quality standards and are used within the same Member State in a hospital under the exclusive professional responsibility of a medical practitioner, in order to comply with an individual medical prescription for a custom-made product for an individual patient are excluded from the scope of this Regulation whilst at the same time ensuring that relevant Community rules related to quality and safety are not undermined. GMP production methodology and - environment remains a requirement, also for these types of (exempted) products. ATMPs, other than tissue engineered products, which were legally on the Community market in accordance with national or Community legislation on 30 December 2008, had to comply with this Regulation no later than 30 December 2011. Tissue engineered products which were legally on the Community market in accordance with national or Community legislation on 30 December 2008 had to comply with this Regulation no later than 30 December 2012. In Belgium, 22 licensed cell- and tissue banks were delivering different types of somatic cell therapy medical products and/or tissue engineered products at the moment the ATMP-transitional period ended. How can hospitals working with engineered cells and tissues on a non-profit basis, for their own patients, cope with the (commercial) ATMP pharmaceutical environment? Are all of the new (pharmaceutical) quality and safety requirements really adding relevant safety and quality to the delivered grafts? How can patients’ access to more than minimally manipulated cell- and tissues be guaranteed? Taking our hospital’s (non-profit) keratinocyte bank (operational since 1987) as an example, we will reflect on what happens when regulators turn the altruistic donation of human body material into commercial products at a certain point in time. Propositions for improvement of regulations will be ventilated. Impact of the issue on tissue processing & inspections will be discussed.

91

O-013 – Import-export and related hot topics S. A. Brubaker

American Association of Tissue Banks (AATB)

Multiple, critical steps are involved to provide human tissues for transplantation that are safe to use and that meet expectations of quality. Due to lack of harmonisation involving “layers of safety,” challenges exist to meet national requirements for import-export of tissue allografts. The AATB continues to address these “layers of safety” by improving professional standards and the accreditation program, and by creating new guidance and educational opportunities for tissue banking professionals. In conjunction with national, international, and state requirements for tissue establishments, these improvements are complimentary and enhance the quality program of a tissue bank. “Hot topics” include: disparities involving donor “consent,” screening and testing; tissue establishment inspections; the evolution of tissue coding and labeling; adequate corrective and preventive action when a deviation is identified; health hazard assessment and recall of tissue; the role of tissue distribution intermediaries; availability of human tissue grafts on the Internet; guidance for identifying, reporting and investigating a tissue recipient adverse reaction; and, the expansion of non-transplant anatomical donation for education.

92

O-014 – Import / export of human tissues in the EU E. Trias

93

O-015 – The emergence of global governance for Medical Products of Human Origin (MPHO) L. Noël

World Health Organisation

In the Public, the complexity of the tissue industry has occasionally led to misunderstanding and misrepresentation fed by lack of transparency and sometimes abusive practices resulting from ignorance, negligence or profit. In too many countries, national health authorities are still unable to account for the origin, the use and the number of tissues procured and use. The regulatory oversight of tissue banking, led by FDA regulations and the EU Directives, is only in project or at the beginning of its development in many countries. In the last decade, non-governmental organizations have been playing an increasing role in the global governance of medical products of human origin (MPHO) beside the influence of advanced national authorities and the role of international regional and global governmental organizations. For instance scientific an professional societies have been at the forefront of the fight against unethical practices in organ donation and transplantation with the Declaration of Istanbul initiated by The Transplantation Society (TTS) and the International Society of Nephrology (ISN). TTS is also engaged in suporting countries in the development of organ donation after death in close collaboration with local professionals and national health authorities WHO. Workshops for the development of suitable hematopoietic stem cells donation and transplantation are also run by the Worldwide Network for Blood & Marrow Transplantation (WBMT). The WBMT promotes global harmonization of all aspects of hematopoietic stem cells donation and transplantation including for instance scientific registries of recipients and monitoring of adverse events and reactions with stem cells donors. Another example of contribution of a NGO to global governance is the development and maintenance by ICCBBA, a non-for profit NGO involving professionals and representatives of health authorities, of the “Information Standard for Blood and Transplant” ISBT128. Indeed, the human origin entails ethical and safety requirements that are common to all MPHO and that professionals and health authorities have to meet to justify the trust of the public and encourage donation. The history of MPHO shows several examples of the magnitude and consequences of transmissible infection. The threat of viruses, prions and of the yet unknown, cannot be ignored. Beyond infections, strict traceability and vigilance and surveillance systems on all severe adverse events and reactions are mandated at national level and across borders when applicable. Reports from vigilance and surveillance systems on severe adverse events and reactions may be very useful to other countries and WHO has been requested by the World Health Assembly to facilitate Member States’ access to such relevant information. The recent cross-WHO initiative on MPHO aims at reaching a global agreement on the exceptional nature of MPHO and bringing back at the forefront the crucial role of the human person and of society for their availability as well as the specific risks associated with the human origin. In particular the project aims at prohibiting financial gain on the human body and its parts as such, in line with the Guiding Principles on Cell, Tissue and Organ Transplantation.

94

O-016 – Information Standard for Blood and Transplant, ISBT 128, the globally endorsed standard for Medical Products of Human Origin (MPHO) R. Warwick

MPHO are donated from humans and have important therapeutic use. They include blood, organs, bone marrow, cord blood, tissues, ophthalmic tissues, reproductive cells and human milk. MPHO provide essential treatment for patients all over the world. Often they cannot be duplicated by other materials. It is critical that these gifts are directed to the specific intended recipient and that they can be verifiably tracked between donor and patient via donation, processing and distribution organisations. Tracking requires appropriate governance within each organisation involved and that the MPHO can be uniquely identified at the global level. It is critical to recognise that tracking includes full traceability throughout the process and at every interim step. Traceability is necessary for safety, vigilance and surveillance, as well as organizational, national and international activity reporting. It is an integral component for recall of MPHO across national and international boundaries to prevent harm to potential recipients of material from a donor or batch in the unusual but real circumstance where there has been an identified adverse event or reaction. ICCBBA is the governing body of the Information Standard for Blood and Transplant, ISBT 128. It works with the international community facilitating the work of volunteers to develop consensus in nomenclature for all sectors of MPHO. ICCBBA has a joint work programme with the World Health Organization (WHO) and has applied to become a Non-Governmental Organization in official relations with the WHO. ISBT 128 is internationally used to support traceability for MPHO in over 4,500 facilities in over 60 countries. It has been endorsed and strongly supported by a wide range of international professional bodies and accreditation organizations and is fully compatible with the Single European Code (SEC). It can be used to provide detailed product information often required by clinicians as well as SEC generic descriptions. Adoption of ISBT 128 by the international tissue community is an important step towards global harmonization and EATB is encouraged to endorse this endeavour.

95

O-017 – Towards the implementation of the single European coding for human tissues and cells intended for human application I. Siska

European Commission, DG SANCO, Brussels, Belgium

According to the European Union (EU) legislation on safety and quality, tissues and cells procured, processed, stored or distributed by EU tissue establishments must be traced from the donor to the recipient and vice-versa. This ability to trace all tissues and cells is essential to ensure rapid reactions when and where safety or quality is at risk. Recognising that developing a coding system to identify tissues and cells used in transplantation is a key tool to ensure full traceability, the EU Directive 2004/23/EC http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2004:102:0048:0058:en:PDF gave mandate to the Commission to design, in collaboration with the Member States, a single European coding system to provide information on the main characteristics and properties of tissues and cells. Following the adoption of the Directive 2006/86/EC http://eur-lex.europa.eu/LexUriServ/site/en/oj/2006/l_294/l_29420061025en00320050.pdf in which the minimum information to be included in the European coding system was laid down, the European Commission organised various activities to further develop this system. After more than five years of hard work and lively discussions, in June 2011 the Member States Competent Authorities unanimously agreed with the structure proposed by their experts in the Coding Working Group and asked the Commission to move to the next steps. The agreed structure of the SEC, which took into consideration the EU heterogeneous landscape in the field of tissue banking (e.g. current practices, the size of the tissue banks, the development of automated systems, etc.), will ensure: (1) a harmonised system for the donation identification in which all EU authorised tissue establishments will have a unique code; (2) flexibility in choosing the product coding system (e.g. national or international), providing that the product codes used at national level are included in the Compendium of tissues and cells products used at EU level; (3) practical implementation by both small and big, multi-tissue banks. Since January 2012 the European Commission is working together with the EUROCET128 www.eurocet128.eu consortium to develop the main tools for the implementation of the single European coding system (SEC): (1) a compendium including all tissue establishments authorised in EU and their respective codes, (2) a compendium with all tissues and cells products manufactured and distributed in EU and their respective codes and (3) the rules and procedures to use the above mentioned codes. In parallel, the European Commission is preparing the legal provisions required to ensure a harmonised use of the SEC across EU. Therefore, the European coding system for human tissues and cells is expected to be operational by the end of 2014. 1 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2004:102:0048:0058:EN:PDF 2 http://eur-lex.europa.eu/LexUriServ/site/en/oj/2006/l_294/l_29420061025en00320050.pdf 3 www.eurocet128.eu/

96

O-018 – Getting ready for implementation of the single European code – information for tissue banks Eurocet128 Consortium: D. Fehily,1 A. Ghirardini,1 P. Ashford,2 R. Benedek,3 F. Vespasiano,1 M. Mareri,1 A. Nanni Costa1 1 Italian National Transplant Centre (CNT) 2 ICCBBA 3 Artman Technologies

The Single European Code for tissues and cells (SEC) was foreseen in Article 10 of Directive 2006/86/EC, and its structure must incorporate the information laid down in Annex VII of that Directive. In 2012, the European Commission awarded a Service Contract (n. 2011 61 02) to the Consortium Eurocet128 to support the implementation of the SEC. The Consortium is composed of 3 organisations:

• The Italian National Transplant Centre (CNT), which maintains the Eurocet Registry;

• ICCBBA, which maintains the ISBT 128 coding standard for human substances, and

• Artman Technologies, a software company. In compliance with the service contract, and with the collaboration of Member State Competent Authorities, the consortium has built and populated the tools to support the application of the SEC:

• A compendium listing the authorised EU Tissue Establishments (TEs) with their authorised activities and their EU codes;

• A compendium including the list of EU tissue/cell product codes and descriptions.

• A translator tool which allows look-up of both compendia and mapping between national/international and EU codes.

At the completion of the service contract, the tools will be hosted and maintained by the European Commission and Member State Competent authorities will be able to access and modify the authorisation status of their Tissue Establishments. The codes listed in the two compendia will allow TEs to construct the Single European Code that must appear on the tissue/cell products distributed by them. A manual for Competent Authorities and Tissue Establishments has been developed to provide detailed instructions on how the code should be allocated to final products. The instructions allow for the continued use of the international system ISBT 128, or existing national systems, with incorporation of the new SEC on the product label. The implementation of the Single European Code will be supported by new or amended legislation.

97

O-019 – Guide to the quality and safety of tissues and cells for human application - Keeping the Council of Europe guidance up-to-date M. López Fraga

European Directorate for the Quality of Medicines & HealthCare (EDQM), Council of Europe, Strasbourg, France

Since 1987, the Council of Europe has contributed actively to the implementation of high standards for the protection of public health and for the promotion of human rights and dignity through its activities in the field of organ, tissue and cell transplantation. The European Committee on Organ Transplantation (CD-P-TO) is the Steering Committee in charge of transplantation activities for the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe). It actively promotes the non-commercialisation of organ and tissue donation, the fight against trafficking in materials of human origin and the development of ethical, quality and safety standards in the field of organ, tissue and cell transplantation. The Council of Europe has just published the 1st edition of the “Guide to the quality and safety

of tissues and cells for human application”. This new guide contains the most up-to-date information and guidance for all professionals involved in donation, banking, transplantation and other clinical applications of these medical products of exceptional nature. As with all transplanted material of human origin, tissues and cells carry risks of disease transmission, which must be controlled by the application of scrupulous donor selection criteria (including testing) and comprehensive quality systems. The idea behind this Guide is to help professionals on a practical level by providing generic guidance that will help improve the rate of successful clinical application of tissues and cells. The Guide makes reference to EU mandatory requirements where appropriate and describes generally-accepted good practice. The Guide has been divided into two parts. Part A contains general requirements applicable to all establishments involved in the donation, procurement, testing, processing, preservation, storage and distribution of tissues and cells. Part B contains specific guidelines and requirements for the different tissue and/or cell types. Applying all of the relevant standards in this Guide will help optimise the quality and minimise the risks of all the complex procedures necessary for tissue and cell donation and transplantation, which will ultimately help improve the rate of successful tissue and cell clinical application.

98

O-020 – Safety, efficacy and quality: Balancing risks and benefits in ATMPs

M. Hildebrandt

TU Munich

Advanced Therapy Medicinal Products (ATMPs) are medicinal products for human use, based on gene therapy, somatic cell therapy or tissue engineering. A rapidly growing area in translational research, they present an area of strategic importance for the European Community. Collectively, these therapies represent the full spectrum of challenges and opportunities of truly disruptive innovation. Some of these therapies need to be integrated speedily and smoothly into existing innovation trajectories, others are set to transcend established marketing and business models in therapeutic medicine.

At the same time, ATMP relying on a highly individualized, labour-intensive therapeutic approach challenge established concepts of regulating medicines innovation. In this field, the central role of academic institutions as drivers not just of the development but also of the manufacture of ATMP has been overlooked. Many products may only reach clinical application by relying exclusively on academic facilities. Compliance with industrial standards in an inherently non-industrial environment creates particular financial, managerial and cultural pressures. Moreover, there is little harmonisation at the level of delivery across the Member States and uncertainty about the regulatory process which, when combined, are stifling academic research and commercialisation of these promising therapies.

On the other hand, public pressure has mounted on several occasions to release unproven therapies in a scenario where due diligence is challenged by patients and their relatives in despair, and jeopardized by third-party interests other than therapeutic benefit, rendering the establishment and defense of sound concepts, proper drug development and effective regulatory control necessary.

For the best of all approaches, drug development, especially in cell-based therapy, will always be guided by balancing risks and benefits. In fact, quality management, risk management and clinical development are intrinsically linked, and have been laid down as consensus documents in current drug regulation.

In my presentation, I will apply this to the field of ATMPs, in conjunction with the understanding of risk and benefit in clinical terms, and I will outline a proposal as to how the field of ATMP Development can be structured and managed to endorse the provision of innovative therapies for patients in need.

99

O-021 – Cleanroom management in cell and tissue banking; the weakest link of the chain is the strongest, because it can break J. Klykens

AC Biobanking – University Hospitals Leuven, Leuven, Belgium

Cleanrooms are used in processing of cell and tissue banking. The cleanrooms have the purpose of limiting the risk of contamination of human body material during processing. A basic risk analysis shows that donor recruitment remains the most important risk in a tissue bank with an established quality management system. This presentation will show how cleanroom management is used at the University Hospitals of Leuven to limit the ultimate risk for patient when they receive an allograft. Although the processing is not considered to be the largest risk in cell and tissue banking, cleanroom management can be used to limit the remaining risk after donor selection. A combination of work methods and bioburden control is able to break the weakest links in the chain, leaving stronger, but separated chains to improve the quality and safety of allografts. The first key element that will break a weak link is the organization of the processing of the material. If we can limit (or eliminate) processing prior to intermediate storage, we are able to evaluate the risk of potential contamination of prelevated tissues and cells and to limit the cross contamination risk by limiting the exposure time. This working method will increase the requirements of bioburden control, which are key to improve quality and safety through cleanroom management. Bioburden control is done through a frequent cleaning plan with low residue detergents and desinfectants. This will have an influence on the monitoring plan of cleanrooms used in cleanroom management. A combined approach of measurements during operation and at rest is used to monitor the performance of the cleanroom. The results of the monitoring plan show positive measurement in a laminar airflow cabinet for 3% of the measurements (>400) and six sigma performance level for the background of these areas. The results show that the cleanroom management is able to reduce bioburden in the cleanrooms and are key elements in risk reduction of cell and tissue banking.

100

O-022 – Analysis of potential factors affecting microbiological cultures in tissue donors during procurement B. Lannau,1 C. Van Geyt,1 G. Van Maele,2 H. Beele1 1 Ghent University Hospital, Ghent, Belgium 2 University of Ghent, 5K3, Ghent, Belgium

Introduction: During the procurement of musculoskeletal grafts contamination may occur. As this might be detrimental for the acceptor, it is important to know which variables influence this occurrence and to alter procurement protocols accordingly. Methods and Techniques: From 2004 to 2012 we gathered information on 6428 allografts obtained from 291 donors. Using a multiple regression model we attempted to determine the factors that influence the contamination risk during procurement. We used the following variables: cause of death, type of hospital (i.e. university hospital versus general hospital), previous blood vessel donation, previous organ donation, donor age, time between death and the start of the procurement, duration of the procurement, number of people attending the procurement and the number of procured grafts. Results: The multiple regression model was only able to explain 5% of the variability of the used outcome variable. Conclusion: None of the variables examined appear to have an important influence on the contamination risk.

101

O-023 – Standardization of tools for tissue engineering M. Doser

102

O-024 – Biobanking: Technologies and quality control A. Leichtle

Sample quality is an essential justification for establishing institutional biobanks as well as a major and non-trivial challenge for their operation. It has to be defined, maintained, and controlled during the whole preanalytical phase of a sample. The main antagonist of sample quality is bias – disturbances and influences we are unaware of, which interfere with the results obtained from the samples in a non-controllable way. Many biomarker studies published in high-ranking journals were based on and could not be replicated due to preanalytical bias. Probably any factor we can imagine could exert effects on the sample quality. Today, analytical platforms might not be sensitive enough to detect the slightest of these alterations – however, biobanking is aimed at collecting and storing samples in larger time frames, and what might seem irrelevant today can render samples useless in a decade. Potential sources of bias are manifold: The patient e.g. is influenced by medication, nutrition, genetic variants, physical activity, gut colonization and many others. The physician interferes with the sample in its retrieval and the many potential differences in the related procedures including the sample transport to the storage facility. The biobank finally accounts for the storage conditions of the sample: temperature, durations, freeze-thaw-cycles, freezing speed, sample preprocessing procedures etc. All of these factors have been identified as relevant, they can be measured and there are ways to minimize their effect. The first and frequently most effective countermeasure against bias is standardization. If all influences and disturbances are kept the same for all samples, they usually will equally interfere with the results of the whole sample set and not with that of a specific subgroup. Another solution, especially for less known and documented sources of bias is randomization. If we spread supposable errors randomly over all samples, it is unlikely that a specific subset gets distorted. The third strategy is exploration with subsequent adaptation of the study/sampling design: if we apply explorative data analysis and find an unexpected grouping, we can search for causative factors and avoid them in subsequent sampling series. However, for all three strategies, thorough documentation is crucial.

103

O-025 – Biobanking: regulatory issues and best practice M. Harder

The storage of tissues and cells at low temperatures is described in numerous scientific articles. The literature discusses example for freezing kinetics, tools and storage conditions. In addition, some organizations and authorities published guidelines that take up the topic and provide valuable information. The presentation discusses these sources and compiles the essential information.

104

O-026 – Banking of cardiovascular allografts: cryopreservation of conventional versus engineered tissues R. Jashari

European Homograft Bank (EHB), International Association, Brussels, Belgium

Cardiovascular allogeneic tissues have been successfully used in clinical practice for more than 50 years in Europe, since the first human aortic valve transplantation by Donald Ross in London in 1962. Basically, the European Directives 2004/23EC, 2006/17EC and 2006/86EC have established rules, which were transposed into the Member State legislation and Standards, specific to their cultural, social and legal needs. Recently, the allograft decellularization has been advocated as the method to improve allograft performance and durability. There are still discussions as to how to consider these tissues, i.e. either as “conventional tissues” or as “ATMP”. In 1975 Marc O’Brien in Brisbane, Australia, introduced cryopreservation as a method for cardiovascular tissue preservation and storage, giving the opportunity to the cardiovascular surgeon to use them for either emergency or elective surgery. Preservation of the cellular structure as well as the matrix was advocated as a main criterion of quality preservation. Finally, parallel to the preserved matrix structure, some level of the viability was necessary to validate the heart valve and/or vessels as appropriate allografts for clinical application. The divergences regarding the immune reaction based on early allograft failure in some patient categories, were for many years very important. In this regard, many clinical and experimental studies have been carried out, showing the controversial final outcome of the allograft valves, depending on age, gender, blood group and allograft size donor/recipient mismatch. Currently, many fundamental research studies have shown, that the immune incompatibility between the donor and the recipient might play an important role in the early allograft failure, particularly in very young patients. Some of the freshly decellularized allografts have been already implanted mainly in the low pressure area (i.e. right ventricular outflow tract) with promising results. In contrast, the data regarding the implantation of those valves in the high pressure area (i.e. left ventricular outflow tract) are still insufficiently inconclusive regarding their long term durability. Can the process of cryopreservation be applied either before or after decellularization for long term storage of decellularized allografts without fragilization of the remaining matrix?- If yes, pediatric cardiac surgeons might offer a long term solution for many new born and young children with severe congenital cardiac malformation. This question must be answered the coming years. A prospective European multi-centric clinical trial “ESPOIR”, supported by the European Commission, regarding the clinical application of decellularized pulmonary and/or aortic valve allografts, might answer this question in the coming years. The cardiovascular tissue banks from the participating countries are invited to support it.

105

O-027 – Banking skin G. Verbeken,1 G. Verween,1 B. Pascual,1 P. De Corte,1 A. Vanderkelen,1 T. Rose,1 S. Jennes,2 J.-P. Draye,1 D. De Vos,1 J.-P. Pirnay1 1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology, Burn Wound Centre,

Queen Astrid Military Hospital, Bruynstraat 1, 1120 Brussels, Belgium 2 Burn Wound Centre, Queen Astrid Military Hospital, Brussels, Belgium

Since 1991, the Queen Astrid Military Hospital (QAMH) exploits a licensed human allogeneic donor skin bank. The QAMH’s Skin Bank is in charge of all process steps, from donor selection to allocation of the harvested skin. The Bank is ISO 9001 certified for the full scope of the process, since 2008. More than thousand donors were released for therapeutic use. The Skin Bank stores viable donor skin in liquid nitrogen tanks but also uses glycerol as a microbiological decontamination tool. Till today, no adverse reactions were reported. The presentation will give a brief overview of the harvesting, storage and distribution protocols that are in use in the QAMH’s Skin Bank today.

106

O-028 – Cornea banking: Today and tomorrow I. Martinache

Agence de la biomédecine (ABM) and CHRU de Lille general Hospital, France

Cornea banking has developed just after world war II in the United States on the blood banks model. Six decades later, preparing, storing, and distributing corneas for allograft is a well-established activity, with about 40000 corneas processed in European tissue banks in 2013, of which about 25000 were distributed for allograft. Corneas benefit from a special status among human tissues. Because non-vascularized and located at the body surface, and its procurement facilitated; because it enjoys a immune privilege when transplanted and the highly symbolic of the sight; because the qualification step at the tissue bank is based on technics of cell density evaluation specific to cornea from one hand; and the storage step in organculture on the other hand , cornea banking remains a dedicated banking, highly represented trough associations such as European Eye Bank Association in Europe. These long time federated professionals and experts in the field provide to the community up to date Technical guidelines for ocular tissue, Minimum medical standards, and others Recommendations fully documented for traditional cornea banking, mainly for penetrating keratoplasty. Standards for quality and safety are also edited in a guide by European commission. This presentation will propose a review of those recommendations and recent literature which set up the state of the art in cornea banking today. Since cornea banking has underwent a first revolution those last years with precutting corneas for lamellar keratoplasty, the way to set up this techniques and its spread remain extremely heterogeneous between European countries. Already, the next revolution seems to merge from acellular matrice and cell therapy, bioingeniering, medical devices progress. It is unlikely that one corneal substitute will be best for all indications, but taken together, the various approaches should probably lead tissue bankers in reviewing techniques, organization and services to fulfill tomorrow’s requests.

107

O-029 – Banking musculoskeletal tissue: which storage temperature is necessary? C. Fölsch

Background: The recommendations for storage temperature of allogeneic bone remain in discussion. No precise scientific data are available to define the accurate temperature. Impact on biological and mechanical properties due to different temperatures have been discussed. An increase of the recommended temperature should reduce costs of bone transplants. Present Recommendations of storage: In the early period of modern bone banking storage at temperature at -80°C was found to be useful. The AATB already recommends storage at -20°C up to 6 months and -40°C for longer lasting storage. Also the EATB has changed recommendations to storage at higher temperatures at -40°C. No adverse effects of lower temperature on the quality of the bone grafts and the clinical outcome have been reported. This includes fresh frozen unprocessed bone grafts which might be of concern regarding possible enzymatic degradation resulting in alteration of biologic behavior within the host. In the beginning of bone transplantation the storage of the transplants was performed at temperatures above -20°C and good clinical results were reported as technical reasons made deeper temperatures impossible since undesired results were often related to bacterial contamination. Conclusion and Outlook: The European guidelines on storage of bone grafts do not recommend exact temperatures. The temperature should be sufficient to retain structure and preserve biomechanical properties of the bone graft. Effects of storage temperature on immunogeneic behavior of the graft have been discussed but no clinical relevant facts could be proved. Different preparations of the bone graft have to be considered regarding the storage temperature. Fresh frozen bone leaves a residual risk of biological effects of different storage temperature on biological behavior since none has been reported on clinical results. Other preparation for example thermodisinfection should exclude the risk of any kind of biological degradation related to the storage temperature since proteolysis should be impossible after the denaturation of the bone graft. The increase of storage temperature up to -20°C should be recommended since no definite adverse effects are obvious. This would reduce costs of bone banking considerably and might increase general availability of hospital bone banks.

108

O-030 – In vivo study on osteoconductivity of supercritical CO2 processed bone allografts, impregnated with tobramycin or vancomycin D. P. Link,1 A. G. Bokhorst,2 K. Kaudela,3 H. Valster1

1 EMCM B.V., Nijmegen, The Netherlands 2 Oude Gracht Group B.V., Den Haag, The Netherlands

3 European Cell and Tissue Bank (ECTB) GmbH, Wels, Austria

Objective(s): Human bone allografts are often used in orthopedic, dental and trauma procedures to reconstruct bone defects. Complications like infections cause serious consequences and requires component removal and thorough local debridement. Usually, long-term systemic antibiotic treatment is required to overcome an infection. Loading bone grafts with prolonged antibiotic release could serve as a local alternative to systemic treatment in case of infections. The aim of this study is to investigate the osteoconductive efficacy of impregnating bone chips with Tobramycin and Vancomycin, in comparison to non-loaded chips. Materials and methods: Bone allografts are processed with supercritical CO2 (scCO2) technique and impregnated with Tobramycin (40 mg/cc bone) or Vancomycin (0,1 gr/ cc bone). On the processus transversus of the spine of 10 Dutch milk goats, validated conduction cages (8 mm Height) were screwed after scraping of the cortex. The chambers of the cages were filled randomly with human scCO2 treated bone chips impregnated either with Vancomycin of Tobramycin, non-loaded scCO2 cancellous bone of different particle sizes, and non-treated, milled goat cancellous chips. The cages were closed with a muscle flap. After 12 weeks the bone chambers were explanted, all samples were embedded and sectioned, and bone formation was analyzed using both light and fluorescence microscopy. Results: Histological analysis revealed new bone formation in all samples, whereby bone had migrated from the transverse process up toward the overlying muscle. No statically significant differences (p>0,05) were measured in the height of ingrowth (>2 and < 6 mm) and the area of new bone formation (>10 and <12% of total area), between the 5 different groups of cancellous bone chips. Conclusion(s): This study shows that impregnation with given doses Vancomycin or Tobramycin of scCO2 treated cancellous bone chips, does not affect the osteoconductive capacity of these allografts, compared to non-impregnated cancellous bone chips.

109

O-031 – Effect of gamma rays (G) and accelerated electron beam (EB) on compact bone collagen damage by assessment of collagen in vitro solubility: influence of defatting, radiation dose and irradiation temperature A. Jastrzebska,1 E. Grazka,1 J. Marowska,1 G. Gut,1,2 I. Uhrynowska-Tyszkiewicz,1,2 A. Kaminski

1,2 1 National Centre for Tissue and Cell Banking, Warsaw, Poland

2 Department of Transplantology and Central Tissue Bank, Medical University of Warsaw, Warsaw, Poland

Introduction and goal: During radiation sterilization of bone tissue grafts, gamma rays or high energy electrons interact with bone collagen, causing both molecular fragmentation of the peptide chains and generation of collagen crosslinks. While collagen fragmentation may adversely affect its mechanical properties and susceptibility to enzymatic degradation, radiation-induced collagen crosslinking can counteract these effects by stabilization of collagen molecules. As the examination of bone collagen solubility in vitro reflects the summary outcome of these processes, we applied this technique in purpose to study the effect of different processing and terminal sterilization methods of compact bone samples on collagen stability. Methods: Compact bone samples isolated from femoral shafts of 6 male donors (aged 46-67 yrs), defatted or non-defatted prior to irradiation, were irradiated with G or EB with two doses (25 or 35 kGy) at different temperature (ambient temperature or dry ice). Non-defatted and non-irradiated bone samples served as control. Following irradiation, bone samples were pulverized under LN2, defatted, and lyophilized. Soluble collagen fractions (Neutral Soluble

Collagen – NSC, and Acid Soluble Collagen – ASC) of bone samples were isolated during 2 successive extractions at 4oC. To measure soluble collagen in NSC and ASC extracts, lyophilized aliquots were hydrolyzed in 6N HCl and hydroxyproline content determined by colorimetric method. Results: Irradiation of compact bone samples by G or EB resulted in tendency to slightly higher solubility of collagen as compared to non-irradiated control samples. The observed tendency seemed to be stronger in bone samples irradiated with EB than in those irradiated with G, and more markedly expressed in samples irradiated with higher dose (35 kGy vs. 25 kGy), especially when gamma rays were applied. No consistent effect of irradiation temperature (ambient temperature vs. dry ice) on bone collagen solubility was found. Similarly, bone samples defatting prior to irradiation appeared not to affect collagen solubility, irrespective of radiation source, as well as irradiation dose and temperature. Conclusion: Radiation sterilization of fresh frozen compact bone tissue grafts seems to have only slight adverse effect on bone collagen stability, with no effect of defatting prior to irradiation – a procedure beneficial for graft biocompatibility.

110

O-032 – MyStem Kit: a non-enzymatic closed system for minimal manipulation of adipose tissue for regenerative medicine applications C. Cicione,1 G. Di Taranto,2 M. Barba,1 M. A. Isgrò,3 E. Di Stasio,3 M. Salgarello,2 F. Michetti,1,4 W. Lattanzi1,4 1 Institute of Anatomy and Cell Biology, Università Cattolica del Sacro Cuore, Rome, Italy 2 Department of Plastic and Reconstructive Surgery, Università Cattolica del Sacro Cuore, Rome, Italy 3 Department of Biochemistry and Clinical Biochemistry, Università Cattolica del Sacro Cuore, Rome, Italy 4 Latium Musculoskeletal Tissue Bank, Rome, Italy

The regeneration and correction of soft tissue volume and defects represent a challenge in aesthetic and plastic surgery. Over the past 20 years, autologous fat grafting (lipofilling) has been extensively used and studied due to its biocompatibility, versatility and low donor site morbidity. To overcome the problem of the low rate of graft survival, micro-fat-harvesting technique and cell-assisted lipotransfer (CAL) has shown the best results. Here, we present an innovative device, named MyStem, based on non-enzymatic tissue separation and cellular enrichment, enabling a ready-to-use micro-harvested fat and a rapid isolation of stromal vascular fraction from human lipoaspirates (SVF). Adipose tissue (AT) from 2 donors was liposucted using the cannula contained in the MyStem Kit. The harvested tissue was alternatively processed using standard protocol (PLA), lipoaspirate fluid protocol (LAF) and MyStem protocol. MyStem protocol consists in the separation of the SVF from liposucted AT in a closed sterile system, allowing washing, filtration and separation of tissue fragments through mesh filters. Tissue morphology was analyzed through histological staining. All isolated cells were comparatively counted, analyzed morphologically, and characterized by flow cytometry for CD45, CD34, CD31, CD105 and CD90 markers. MyStem protocol required 5-10 minutes. The harvested AT comprised small lobules, with intact cell membranes and structurally integer adipocytes. histology showed the presence of cells without lipid content and placed in clusters at the axis of the stromal lobule. The number of cells/gr isolated was respectively: 4x105 PLA, 1.8x105 LAF and 2x105 MyStem. Cells isolated with the three alternative protocols displayed a spindle shaped, bipolar morphology, upon seven days of culture. Flow cytometry revealed a comparable expression of surface antigens between the three populations of cells. These results provided the first proof of principle on the use of MyStem kit to collect intact AT and separate a cell mixture enriched with ASC from a lipoaspirate sample. The micro-harvested AT could be used alone or in combination with the cell-mixture isolated within the same protocol. MyStem products have the characteristic of a minimally manipulated tissue that can be used in regenerative medicine.

111

O-033 – Minimization of allogeneic tissue immunogenicity by cryopreservation K. G. M. Brockbank,1,2,3 L. H. Campbell,1 Z. Chen,1 E. D. Greene,1 U. A. Stock,4 M. Seifert5 1 Cell & Tissue Systems, Inc., North Charleston, SC, USA 2 Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA

3 Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, SC, USA

4 University Hospital Frankfurt, Dept. Thoracic, Cardiac and Vascular Surgery, Frankfurt am Main, Germany

5 Institute of Medical Immunology and Berlin-Brandenburg Center for Regenerative Therapies (BCRT),

Charité Universitätsmedizin, Berlin, Germany

Objective: Allogeneic tissue engineered tissues potentially impact huge cardiovascular and orthopedic graft applications. We have previously demonstrated in a large animal model that ice-free cryopreservation of pulmonary heart valves employing high concentrations of cryoprotectants reduces T-lymphocyte infiltration and improves in vivo outcome compared with traditional dimethylsulfoxide cryopreservation by freezing. Here we report experiments to extend prior large animal heart valve studies to engineered human tissues in vitro to determine whether or not A) human peripheral blood mononuclear cells (hPBMCs) respond to ice-free cryopreserved tissues and B) whether residual cryoprotectants may be cytotoxic for the graft recipient’s cells after implantation thus creating a false impression of decreased immunogenicity. Methods: Grafts were generated in the laboratory by culturing human smooth muscle cells on a polyglycolic acid scaffold. Experimental 4-5mm human engineered blood vessels (TEBVs) samples were cryopreserved with an 83% formulation cryoprotectant (DMSO, propanediol and formamide) by simply placing and storing samples in cryoprotectant solution at -135°C. The cryoprotectants were removed using three washes. Fresh and cryopreserved TEBV samples were compared employing hPBMCs co-cultures for assessment of proliferation and cytokine release in vitro. Unstimulated and phytohemagglutinin (PHA) stimulated hPBMCs were employed as negative and positive controls. Proliferation was assessed using the resazurin assay and cytokines (IFNγ, TNFα, IL-2, IL-6, IL-10, IL-12 and IL-18) using ELISA kits. Decellularized and cryopreserved TEBVs were compared using human umbilical vein endothelial cells (HUVECs) in vitro to determine whether residual cryoprotectants in cryopreserved TEBVs reduced HUVEC proliferation. Decellularized tissues were made using enzymes and detergents leaving an extracellular matrix tube and refrigerated in PBS until testing.Cell viability and survival over a week of cell culture was assessed using the resazurin reduction assay after which the constructs were fixed in neutral buffered formalin, sectioned and stained. The results were compared employing a t-test and one way ANOVA. Results: The results demonstrate that there was no stimulation of hPBMC proliferation and significantly reduced IL6, IFNγ, TNFα, and IL-10 production by cryopreserved TEBVs compared with fresh untreated TEBVs (p<0.05). Fresh TEBVs also stimulated a 2-fold increase in hPBMC proliferation compared with cells only controls, similar to PHA stimulated positive controls. No stimulation of IL-2, IL-12 or IL-18 was observed. There were no statistically significant viability differences comparing HUVEC seeded decellularized and cryopreserved TEBVs after 2, 4 or 8 washes in cell culture. The HUVECs formed a confluent monolayer on all surfaces of the cryopreserved TEBVs, regardless of the number of post-rewarming washes, demonstrating that any residual cryoprotectants present were not cytotoxic. Conclusions: We conclude that ice-free cryopreservation of allogeneic TEBVs significantly reduces immunogenicity and that the results are not confounded by cytotoxicity due to residual

112

cryoprotectants. In parallel experiments, similar results were obtained in a xenogeneic porcine heart valve leaflet model with CD3 co-stimulation and hPBMCs. Our working hypothesis is that “the new cryopreservation method is modifying or masking tissue signals perceived by responder cells”, most likely damage-associated molecular pattern molecules (DAMPs) that can initiate and perpetuate immune responses. Clinical partners are being sought for patient tissue implantation trials. This work was supported by NIH Grant #R43EB014614.

113

O-034 – Mesenchymal stem cell immunosuppressive therapy Y. Beguin

Dpt of Hematology & Laboratory of Cell and Gene Therapy (LTCG), CHU of Liège, Liège, Belgium Laboratory of hematology, GIGA-I3, GIGA Research Institute, University of Liège, Liège, Belgium

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for many patients with hematological malignancies. Allo-HCT may be complicated by the occurrence of graft-versus-host disease (GVHD), consisting in the destruction of recipient organs by immune cells contained in the graft. GVHD may occur within 3 months of transplantation (acute GVHD) or later (chronic GVHD). Alloreactivity of donor immunocompetent cells in the graft against the host tumor (graft-versus-tumor (GVT) effect) also plays a major role in eradicating malignancies after allo-HCT. This prompted the development of allo-HCT with non-myeloablative conditioning in which the burden for tumor eradication has been shifted from high-dose chemo-radiotherapy towards GVT effects. Such minitransplants are characterized by gradual replacement of recipient by donor-derived hematopoiesis and immune system. However, graft rejection rates are increased compared to conventional transplants. GVHD, while a little less frequent than in conventional transplants, remains the leading cause of non-relapse mortality.

Mesenchymal stem cells (MSC) are multipotent progenitors within the bone marrow (BM) capable of differentiating into various cells and tissues, such as chondrocytes, osteoblasts and adipocytes. MSC suppress lymphocyte proliferation and inhibit naive and memory T-cell responses to their cognate antigens in vitro. This appears to be independent of the major histocompatibility complex, as inhibition is similar using autologous or 'third party' MSC. Modulation of T-cell activation and immune suppression by MSC involves soluble factors such as TGF-β, HGF, bone morphogenic protein 2, or PGE2 and induction of regulatory T cells (Treg). MSCs also exhibit immunosuppressive properties in vivo, such as prolonging allogeneic skin graft survival or preventing experimental auto-immune encephalitis. Immunodeficient mouse models have also shown that co-infusion of MSC can facilitate engraftment of cord blood or marrow HSC.

We have developed a standardized protocol for MSC expansion. Thirty ml of bone marrow are collected under local anesthesia from screened healthy volunteer donors. Mononuclear cells are cultured in sterile flasks in the presence of gamma-irradiated fetal bovine serum for about 4 weeks, after which MSC are harvested and frozen at -192°C. Quality controls include cell counts and viability, immunophenotype, karyotype, extensive microbiological testing (bacterial and fungal cultures, mycoplasma and endotoxin) and potency testing (multilineage cell differentiation and immunosuppressive properties). A bank of clinical-grade MSC available for clinical trials has thus been set up. MSC do not need to be HLA-matched with the patient and can be injected intravenously without significant side effects.

We have then set up a number of academic clinical trials using these banked MSC, both at the CHU of Liège and in the collaborative setting of the Transplantation Committee of the Belgian Haematological Society, after approval by the AFMPS/FAGG and EC.

1. Treatment of acute GVHD : first line treatment for acute GVHD consists in high-dose steroids that produce sustained responses in only 25-40% of patients, outcome being dismal for non-responding patients. Le Blanc et al. and other small studies have demonstrated that infusion of third party MSC could be successfully used as treatment of severe, steroid-refractory, acute GVHD. We aimed at investigating this in a Belgian multicenter setting.

114

2. Treatment of poor graft function (PGF) : PGF occurs in 5-25% of patients after allo-HCT and is associated with consequent morbidity and mortality due to infections and hemorrhagic complications. A small study has suggested that MSC might help improving hematopoiesis in this setting. We aimed at investigating this in a large number of patients in a Belgian multicenter trial.

3. Treatment of low donor T-cell chimerism after allo-HCT : low T-cell chimerism is associated with a high risk of graft rejection after nonmyeloablative HCT and donor lymphocyte infusions (DLI) are not very efficient. MSC given before DLI might help to prevent graft rejection by decreasing host-versus-graft reactions. We are investigating this in a Belgian multicenter setting.

4. Prevention of GVHD and graft rejection : we are conducting 3 clinical trials in which we evaluate the potential of a co-infusion of MSC on the day of HCT to prevent GVHD and graft rejection :

- In the context of cord blood transplantation : Belgian multicenter trial

- In the context of myeloablative transplantation from unrelated donors : 2-center trial with K Leblanc (Karolinska Institute)

- In the context of non-myeloablative transplantation from mismatched donors : we have published a first pilot study in 20 patients compared to a group of 16 patients not receiving MSC (BBMT 2010). There was a trend for less acute and chronic GVHD in the MSC group, and 1-yr OS and PFS were 80% and 60%, in this group, compared with 44% (P=0.02) and 38% (P=0.2) in the control group, respectively. We are now conducting a Belgian, multicenter, placebo-controlled trial in 120 patients to confirm these encouraging results.

5. Prevention of graft rejection after liver or kidney transplantation : we are conducting a pilot trial at the CHU of Liège, using a single infusion of MSC to help prevent graft rejection, and possibly totally withdraw immunosuppression after liver transplantation.

6. Treatment of refractory Crohn’s disease : a small study has suggested that some patients with refractory Crohn’s disease might respond to MSC therapy. We are now conducting a pilot study to assess safety and efficacy of 2 consecutive MSC infusions in 20 patients with Crohn’s disease refractory or intolerant to conventional therapies including infliximab.

115

O-035 – The Cardiac Atrial Appendage Stem Cell: a new candidate for myocardial repair J.-L. Rummens,1,2 R. Koninckx,1,2 A. Daniels,1 S. Windmolders,1,2 U. Mees,3 R. Macianskiene,4 K. Mubagwa,5 P. Steels,2 L. Jamaer,6 J. Dubois,6 B. Robic,2,3 M. Hendrikx,2,3 K. Hensen1,2 1 Laboratory of Experimental Haematology, Jessa Hospital, 3500 Hasselt, Belgium 2 Faculty of Medicine and life sciences, Hasselt University, 3500 Hasselt, Belgium 3 Department of Cardiothoracic Surgery, Jessa Hospital, 3500 Hasselt, Belgium 4 Laboratory of Membrane Biophysics, Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania 5 Department of Cardiovascular Diseases, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

6 Department of Cardiac Anaesthesia, Jessa Hospital, 3500 Hasselt, Belgium

Stem cell therapy is a novel approach to restore cardiac function after myocardial infarction (MI). Over the last decade, the results of clinical trials involving either intracoronary or intramyocardial transplant of bone marrow (BM)-derived stem cells have been published. The minor improvement of cardiac function reported may be explained by the limited cardiomyogenic differentiation potential of BM-derived stem cells. Nevertheless, transplantation of BM-derived mesenchymal stem cells (MSCs) can be beneficial because it can promote the survival of cardiomyocytes (CMs) through paracrine effects. To develop more successful stem-cell therapies, the scientific focus has shifted from BM-derived stem cells to cardiac stem cells (CSCs) because they are probably programmed to become CMs. The presence of a multipotent endogenous c-kit+ CSC population was first described in rats and later in humans. Since then, several groups have reported the isolation of CSCs expressing a variety of other markers or functional properties, e.g. islet-1, Sca-1, cardiac side population cells, and cardiosphere-derived cells (CDCs). Despite initial promising in vitro and pre-clinical research data, the first reported clinical trial with CDCs demonstrated no significant change in cardiac function, making further investigation necessary. A promising strategy for purifying viable stem cells from tissues is based on the aldehyde dehydrogenase (ALDH) enzymatic reaction mechanism. High ALDH activity has already been attributed an important feature of several stem-cell types including mesenchymal, neural and recently also cancer stem cells. By using ALDH as an isolation marker, we identified a new ALDH+CD34+CD45- stem cell population in human atrial appendages with superior cardiomyogenic differentiation capacity: the cardiac atrial appendage stem cells (CASCs). CASCs possess a unique phenotype that is clearly different from c-kit+ CSCs but that seems more related to the recently described cardiac colony-forming-unit fibroblasts. Based on immunophenotype and in vitro differentiation studies, we suggest that CASCs are an intrinsic stem cell population and are not mobilized from bone marrow or peripheral blood. Indeed, they possess a clonogenicity of 16% and express pluripotency-associated genes. Furthermore, compared with cardiosphere-derived cells, CASCs possess an enhanced cardiac differentiation capacity. Differentiated cells express the most important cardiac-specific genes, produce troponin T proteins, and have an electrophysiological behaviour similar to that of adult cardiomyocytes (CMs). Transplanting CASCs in the minipig MI model resulted in extensive cardiomyogenic differentiation without teratoma formation. The identification of this new CASC population in human heart tissue opens interesting perspectives for cell therapy in patients with ischaemic heart disease.

116

O-036 – Limbal epithelial stem cell culture: an ongoing effort M.-J. Tassignon,1 N. Zakaria,1,2 T. Possemiers,1,2 I. Leysen,1 C. Koppen1 1 Ophthalmology, Antwerp University Hospital, Antwerp, Belgium

2 Centre for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Antwerp, Belgium

Purpose: To describe the results of a phase I/II clinical trial for standardized, non-xenogenic, cultivation and “no touch” surgical transplantation of limbal stem cell grafts. Methods: 18 eyes of 18 patients were transplanted with either autologous (n=15) or allogenic (n=3) limbo-amnion composite grafts that were generated using a standardized culture protocol free of xenogenic culture products and transplanted using a standardized “no touch” surgical technique. In vitro cellular outgrowth and phenotype of the limbo-amnion composite graft was assessed prior to transplantation. The clinical outcome measures investigated were: corneal neovascularization, central corneal opacity, pain, photophobia and visual acuity pre and post transplantation. Results: Limbal epithelial cells showed an average outgrowth of 14.2mm ±3.7mm by day 14. The majority of the cells displayed a progenitor phenotype: p63 bright, CK14, ABCG2 bright and desmoglein, CK3/12 dim protein expression. The transplant recipients were followed up for a mean of 22 months (range 4-43 months). 12 out of the 18 transplant recipients were graded successful (12 had anatomical success and 7 also attained some degree of functional success), giving an overall success rate of 67%. We did not see a significant reduction in pain, photophobia or central corneal opacity for the patient group post transplant. However, the ocular surface photographs for pre- and post stem cell transplantation, showed a significant (p=0.007) reduction in the percentage area of corneal neovascularization [Fig.1]. Conclusions: We have been able to show that our standardized, xenogenic free culture system and “no touch” surgical technique has outcome measures comparable to other clinical studies. This technique has the added advantage of being free from animal contaminants such as mouse feeder layers and foetal bovine serum. Improved functional success is attained once penetrating keratoplasty is performed following successful stem cell grafting.

117

Fig 1. Eyes with total limbal stem cell deficiency before (A, E) & after (C, G) limbal stem cell transplantation within the software program for corneal neovascularization (CNV) mapping: (B, F, D, H). There was a significant reduction in % area of CNV post limbal stem cell transplantation (I) (**p= 0.007), but no significant decrease in the degree of corneal opacification post stem cell transplant (J).

118

O-037 – Qualitative and quantitative differences of adipose tissue-derived stem cells from superficial and deep subcutaneous lipoaspirates: a matter of fat W. Lattanzi,1,2 G. Di Taranto,1,3 C. Cicione,1 M. A. Isgrò,4 M. Barba,1 G. Visconti,3 E. Di Stasio,4 M. Salgarello,3 F. Michetti

1,2 1 Institute of Anatomy and Cell Biology, Università Cattolica del Sacro Cuore, Rome, Italy

2 Latium Musculoskeletal Tissue Bank, Rome, Italy

3 Department of Plastic and Reconstructive Surgery, University Hospital "A. Gemelli", Università Cattolica

del Sacro Cuore, Rome, Italy 4 Institute of Biochemistry and Clinical Biochemistry, Università Cattolica del Sacro Cuore, Rome, Italy

Fat grafting is a common reconstructive procedure with several clinical applications; much effort have been spent during the last decades in investigating the biology behind the success of this procedure. Adipose tissue is indeed a valuable source of somatic stem cells, mainly of mesenchymal lineage, endowed with attractive trophic features and potential for tissue regeneration. This made fat extremely attractive for regenerative medicine approaches, based on the wide availability and the ease of retrieval. The aim of this study is to comparatively characterize different cell populations in human superficial and deep adipose tissue obtained by lipoaspiration in three different regions (thigh, knee, abdomen). Superficial and deep adipose tissue specimens from the three regions were collected through liposuction from 10 female patients (aged 34-to-48 year old). All samples underwent: 1. histological examination, 2. separation of the stromal vascular fraction and further cell isolation through centrifugation and enzymatic digestion, 3. FACS analysis of naïve cells a 4. Expression analysis of adipogenic markers by real time PCR. Adipose tissue structure was adequately preserved in all specimens; the cell immunophenotype indicated a higher amount of CD105+ stem cells and of CD34+ angiogenic cells in the superficial tissue as compared to the deep one. Cells were negative for CD31 and CD45 in all samples, as expected. In particular, a significantly higher amount of CD105+ and CD34+ cells was found in abdomen and thigh samples. The expression of all tested markers of adipocyte differentiation, namely S100B protein and its receptor RAGE, adiponectin and its receptor ADIPOQR1, was significantly increased in the deep fat compared to superficial specimens. Taken together, the present data suggest that superficial subcutaneous fat should contain somatic cells in a “less differentiated” status, thus proving a higher ‘stemness’ profile. This makes this tissue the best candidate for fat grafting aimed at successful tissue regeneration approaches.

119

O-038 – Customized bone allografts for complex defect reconstruction: the use of computer numerically controlled milling T. Schubert,1 M. Verhelpen,1 S. Selvais,1 B. Godts,1 P. Lejuste,2 C. Raftopoulos,3 D. Dufrane1 1 Unit of Tissular and Cellular Therapy of the Locomotor Apparatus, Center of Tissue and Cell Therapy, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium 2 Department of Maxillo-Facial Surgery, Clinique Sainte-Elisabeth, Namur, Belgium

3 Department of neurosurgery, Cliniques Universitaires Saint-Luc, Université catholique de Louvain,

Brussels, Belgium

Introduction: In bone tissue reconstruction, computer-aided design and manufacture (CAD/CAM) is an efficient tool to create patient-specific implants (PSIs). Human bone allografts are often used for complex bone tissue reconstruction. However, to shape the allograft to the required dimension is a delicate and time consuming procedure. Therefore, we proposed a CAD/CAM design for a computer numerically controlled (CNC) milling of a PSI from a treated bone allograft. Goal: to assess the effects of a CNC milling on five different sources of bone allografts, select the best bone source for this application and evaluate the feasibility of a clinical application of this procedure. Material and methods: Heat generation (thermal imaging), density modifications (peripheral quantitative computed tomography), surface alteration (scanning electron microscope) and cellular adhesion (24h static cell culture using adipose mesenchymal stem cells) were assessed during milling of bone from five sources (femoral head, great trochanter, lumbar vertebrae, talus and calcaneum). Based on the experimental results, talar bone was used to create three types of custom spine fusion cages (n = 24) and 28 maxillomandibular PSIs for neurosurgery and maxillofacial surgery, respectively. Results: Temperature elevation during the milling procedure remained very limited (mean 34.4 ± 7.2°C for all groups, p = NS between all groups). The milled surface appeared rough and shredded with cutting debris clogging the bone pores. Cellular adhesion was unaffected by bone type (<4% at 24 h of incubation). The talus demonstrated the highest pre- and post-milling density (285.6 ± 22.2 and 307.2 ± 14.9 mg/cm3, respectively) compared to all other groups (p < 0.05), and the most isotropic repartition in trabecular bone. Density and temperature generation were significantly correlated in the talus (Pearson’s r = 0.702, p < 0.001). In the clinical setup, the use of these PSIs proved easy to implement. Histological samples of one maxillofacial implant demonstrated full incorporation and cellular recolonization of the graft at 1 year post-implantation. Conclusion: CNC milling is a reliable and effective procedure to precisely shape a bone allograft for specific surgical demands and complex bone reconstructions.

120

O-039 – Comparison of different stem cell populations for vascularization and integration of an acellular human dermis graft seeded with autologous cells for standardized defect wounds M. Vitacolonna,1 M. Smith,2 E. Rößner,1 P. Hohenberger1 1 Division of Surgical Oncology and Thoracic Surgery, Department of Surgery, University Medical Centre

Mannheim, Mannheim, Germany 2 German Institute for Cell and Tissue Replacement, Berlin, Germany Introduction: In irradiated tissue, wound healing can be substantial impaired. The transplantation of an intact human acellular dermis (hAD) seeded with autologous cells could theoretically lead to an improvement in wound healing. However, cells may be damaged due to initial ischemia after transplantation. The co-culturing of adult stem cells might improve the vessel formation inside the implanted matrix. To compare the in-vivo vascularization potential of adult stem cells, mesenchymal stem cells (MSC), endothelial progenitor cells (EPC) and the unseparated cell mixtures of stromal-vascular fraction (SVF) of adipose tissue and bone marrow (BMDC) were examined by intravital microscopy. Matrices seeded with fibroblasts and pericytes were used as control group. Materials and methods: Autologous cells were isolated from rat bone marrow and adipose tissue, characterized and cultured. 72 rats received a dorsal skinfold chamber and were divided into 2 main groups, irradiated and unirradiated. Rats were irradiated with 20Gy. Each of these 2 groups were further subdivided into 6 groups with n=6 rats. Matrices (5x5 mm) were seeded

dynamically on both sides with each 5x105 cells/hAD. Implanted scaffolds were examined every 3 days for a total of 12 days. Vessel densities were quantified semi-automatically. Results: In general, irradiated animals showed a significantly lower vascularization than the non-irradiated rats. In the unirradiated groups only the SVF group achieved a significantly higher vessel density at day 6 compared to the control group. However, the MSC and SVF groups showed at the 3rd postoperative day a weak vessel ingrowth. In the irradiated groups a significantly higher vascular density could be achieved by the MSC and SVF, but only in the SVF group vessel ingrowth was visible at the 3rd day. Discussion: It has been shown that in both main groups a significantly higher vascular density has been achieved by the uncultured heterogen SVF compared to the groups seeded with fibroblasts. In addition to the simple and immediate availability compared to adult stem cells, the use of the SVF can avoid the loss of pluripotency and spontaneous immortalization caused by long cultivation.

121

O-040 – Tissue engineered biovital human skin substitute W. Łabuś,1 M. Kawecki,1,2 A. Klama-Baryła,1 D. Hoff-Lenczewska,1 M. Kraut,1 M. Maj,1 J. Glik,1,2 M. Nowak1 1 Centre For Burn Treatment in Siemianowice Śląskie, Poland

2 Department of Health Science, University of Technology and Humanities in Bielsko Biała, Poland

Introduction: The process of removing cells from human allogenic dermis gives the opportunity to obtain acellular, collagenous, non-immunogenic dermal matrix. Such a biomaterial can be in

vitro revitalized by autologous cells. Goal: To point out an optimal method of cell removal from human dermis. To point out an optimal method of production of human living skin substitute based on obtained acellular dermal matrix. Method: The proposed research program has a positive opinion of the ethics committee. Research material was sterilized by ionizing radiation with a dose of 35kGy.The samples underwent three different decellularization procedures. First group was treated with proteolitic enzymes, second group was treated with detergent and the third group was prepared by mixed two-step method based on the use of proteolytic enzyme and detergent. Evaluation of the results of the experiments were examined histologically, and the evaluation of the structure of the extracellular matrix at the nano scale were performed using an atomic force microscope. Next, the bioprosthesis were in vitro repopulated with fibroblasts. The results of this experiment were examined histologically. Result: The results of the decellularization processes of the human dermis are shown in the atomic force microscopy images. The nanostructure of the dermal extracellular matrix was examined and dimensioned .The results of the decellularization processes of the human dermis are shown in the histological images. In the histological images the degree of eradication of unwanted cellular debris and the appearance of connective tissue fibers were examined. The results of the in vitro fibroblast culture on the acellular dermal matrices, the number and viability of the cells were analysed with use of cytometer. The results of the revitalization of the acelullar dermal matrices were verified histologically. On the basis of histological images the degree of fibroblasts occupancy was examined. Conclusion: The most optimal method for removing cells from human dermis is a mixed two-step method based on the use of proteolytic enzyme and detergent. The most optimal method of production of living human skin substitute is the procedure based on the in vitro culture of fibroblasts on the acellular dermal matrix obtained using the enzymatic method. This work was supported by the National Centre for Research and Development, project POLYCELL PBS1/B9/10/2012.

122

O-041 – Regeneration of ischemic muscle with a composite graft made of adipose mesenchymal stem cells or bone marrow mesenchymal stem cells and human acellular collagen matrix M. van Steenberghe,1 T. Schubert,1 R. M. Goebbels,1 C. Galli,2 D. Dufrane1 1 Cellular and Tissular Therapy Center, Cliniques Universitaires Saint-Luc, Brussels, Belgium

2 Laboratorio di Tecnologia della Riproduzione, University of Bologna, Bologna, Italy

Introduction and Goal: This study aims to compare, both in vitro/in vivo, the efficacy of a composite graft made of Mesenchymal Stem Cells (derived from Bone marrow, BM-MSC and adipose tissues, AMSCs) and a human acellular collagen matrix (HACM) to promote the regeneration of ischemic muscles. Materials/Methods: Porcine BM-MSCs/AMSCs were in vitro compared to assess the cellular viability/growth factors release (VEGF,FGF,HGF,IGF,TGF) under normoxic/hypoxic conditions at 0.1/21 % Oxygen for 72h, respectively. The capacity of MSCs adhesion on the HACM was investigated by confocal microscopy up to day 30 post-incubation. Two in vivo models (nude rats, n=24) were designed to study the muscular reconstruction: (i) Model 1: full thickness abdominal wall defect cured with HACM alone/AMSCs or BM-MSCs+HACM (assessed at day 30 post-implantation); (ii) Model 2: muscular necrosis induced by electrocoagulation (4 necrosis areas/recipient) treated with HACM alone/AMSCs or BM-MSCs+HACM and Sham procedure (no treatment). In this latest model, the capacity regeneration was studied at day 15/30 post-implantation to investigate the angiogenesis promotion/necrotic thickness remodeling. Histomorphometry was performed to assess matrix remodeling (HE/dystrophin), revascularization (Masson’s Trichrom) and MSCs tracking by an anti-Green Fluorescent Protein (GFP) immunostaining. Results: No significant difference was found to obtain 90% of HACM recovering by both BM-/AMSCs at day 18 post-incubation. A significantly lower secretion of IGF was observed for AMSCs in comparison to BM-MSCs in hypoxic conditions (-97.6%, p<0.005). In contrast, a significantly higher VEGF/FGF secretions was found for AMSCs in comparison to BM-MSCs (+92%, p<0.001 and +72%, p<0.05, respectively). In both models, the angiogenesis was significantly higher in treated areas by AMSCs+HACM in comparison to BMSCs+HACM and HACM alone (Model 1: 434±11% vs. 215±1%/145±6%, p<0.05, respectively; Model 2: 21±1% vs. 11±2/12±3, p<0.001, respectively). In Model 2, a significant decrease of necrotic thickness ratio was found with AMSCs or BM-MSCs +HACM in comparison to HACM alone (23±9% and 24±10% vs. 49±17, p<0.05,respectively). After GFP immunostaining, both MSCs were found to remain undifferentiated without any co-staining for dystrophin. Conclusion: A composite graft of MSCs and HACM can promote remodeling of muscular defect through the paracrine effect of undifferentiated MSCs. Moreover, adipose tissue appears to be a better source for ischemic soft tissue regeneration.

123

O-042 – Different methods of cell viability measurement applied on CBU-attached segments: a comparative study of their predictive value T. Devos,1 F. Sinap,1 J. Klykens,1 A. Laenen,2 H. Schoemans,1 M. Boogaerts1 1 Cord Blood Bank Leuven (Leuvense Navelstrengbloedbank), UZ Leuven, Belgium 2

Interuniversity Centre for Biostatistics and Statistical Bioinformatics, KU Leuven, Belgium

Objectives: According to Netcord-FACT standards, reference samples stored in identical conditions as the cord blood unit (CBU) are required for viability and potency measurement before release for transplantation. In our cord blood bank, we perform both 7-AAD and trypan blue staining (TB) for WBC viability and 7-AAD for CD34 viability on an attached segment. We explored the predictive value of both methods of cell viability measurement, differences between viability in CBU and segment and time-dependent decline of viability during these measurements. Methods: Total WBC viability was measured by both TB and 7-AAD in 14 thawed segments (SEG) and CBU. Samples were representative of different storage times. TB was measured immediately (TB0) and 30 min after thawing (TB30) in both SEG and CBU. 7-AAD was performed to assess viability of WBC and CD34. CFU were plated for both SEG and CBU. Results: For both 7-AAD and TB0, significantly lower WBC viabilities were measured in SEG compared to CBU (-18.2%(p=0.0001) and -10.4% (p=0.0024 respectively)). This was also true for CD34 viability measured by 7-AAD (-7.0% (p=0.0333)). Although the systematic difference in WBC viability was larger in case of 7-AAD compared to TB0, a positive correlation was found between SEG and CBU using 7-ADD for both WBC and CD34 cell viability (Pearson r=0.60; p=0.023 and Pearson r=0.72; p=0.004). No significant correlation existed with TB0 (r=-0.09; p= 0.74). Intrinsic to the 7-AAD technique, cells are stained only after 30 minutes. When TB was applied after 30 minutes, mean viability in SEG decreased from 71 to 48 %. The mean viability in the segment with 7-AAD was 60 %. All SEG and CBU showed excellent clonogenicity. Conclusions: For both 7-AAD and TB, total WBC viability is lower in the segment compared to the CBU. Although the systematic difference in viability between segment and CBU was larger with 7-AAD compared to TB, the former technique has a better predictive value of viability. Rapid staining of cells with TB is crucial. One of the reasons for systematically lower viabilities of 7-AAD compared to TB could be the delay of cell staining intrinsic to the 7-AAD technique itself.

124

O-043 – Beta Cell therapy for diabetes D. Pipeleers

Brussels Free University-VUB

The pancreatic beta cell population maintains blood glucose control through physiologic regulation of its insulin synthesis, storage and release. This homeostatic role requires a sufficient number of beta cells and an adequate functional state of the cells, collectively defined as

functional beta cell mass. In type 1 diabetes, the beta cell number is severely reduced and residual beta cells are impaired, often leading to complete loss of metabolic control. Implantation of an adequate functional beta cell mass could restore this deficit and is therefore considered as treatment of choice for this disease. Methods have been developed for isolating and culturing beta cell-enriched preparations from human donor pancreases, and for standardizing their composition and function according to quality control criteria. Their intraportal transplantation in type 1 patients was shown to reinstall endogenous control of glycemia, significantly reducing its fluctuations, also in patients who could not stop insulin injections. In most recipients this effect progressively declines during the following years. An insufficient functional beta cell mass at start is a major cause for this decline. In addition, shortage in human donor organs seriously limits further development of this therapy into a durable and widely used application. Large-scale production of grafts from alternative

sources could solve the quantitative limitation and at the same time provide preparations according to quality control standards. We undertake preclinical studies with purified porcine beta cell grafts (provided by Beta-Cell nv, Belgium) and with beta cell precursor grafts derived from human embryonic stem cells (provided by ViaCyte-Inc, US). These novel cell therapy products are tested under encapsulated form in order to protect them against inflammatory and immune reactivity. The data will drive translation to clinical trials. Our program illustrates the need and potential of a tight collaboration between university (hospital) departments and bio industry. It also requires a commitment of society to facilitate development of cell therapy in clinics in the perspective of curing serious diseases.

125

O-044 – Active specific immunotherapy for High Grade Glioma

S. Van Gool, N. Ectors, S. De Vleeschouwer

KU Leuven, Belgium

High grade glioma (HGG) is an orphan cancer disease with a dismal prognosis in spite of neurosurgery, radio- and chemotherapy. Several groups demonstrated that immunotherapy could be used as an innovative treatment. Most groups use autologous monocyte-derived dendritic cells (DCs) loaded with tumor antigens. We have set-up a translational research program and focus on mature DCs loaded with antigens derived from tumor lysate (DCm-HGG-L). The methodology has been worked-out pre-clinically, and we have developed several research programs to improve efficacy. We translated the methodology into clinical practice for patients with relapsed HGG (HGG-IMMUNO -2003 cohort comparison study) and patients with primary diagnosis of grade-IV HGG (pilot trial, HGG-2006 trial and the currently recruiting prospective double-blind placebo-controlled phase IIb randomized clinical trial HGG-2010 study). Induction vaccines consist of 4 weekly injections with DCm-HGG-L, while boost vaccines consist of HGG-L. The clinical program recruits patients from 19 neuro-oncologic centres in Belgium and from 26 countries outside Belgium. Annually about 100 new patients enter into the program. The production of DCs and tumor lysate is feasible. The elaborated quality control system is patient-oriented because of the mix of GTP and GMP for multiple individual vaccine productions per patient to be administered as part of multimodal primary treatment approaches within GCP and with materials derived from individual very ill patients who recently underwent major neurosurgery, intensive care activities and corticosteroids all affecting the leukapheresis product. The progression-free and overall survival of the patients significantly depend on the recursive portioning analysis (RPA-IMMUNO) using risk factors like age, grade of histology, Karnofski performance scale and postoperative mental status. With RPA we demonstrated step-wise improvements in prognosis by improving the technology of the vaccine production. Moreover we demonstrated with the HGG-2006 data which subgroup of patients clearly benefit from vaccination as compared to historical control patients classified according to the EORTC-RPA. The latter made it possible to define the randomization stratification of the HGG-2010 trial. We propose a concept of Advanced Therapy Treatment to complement the existing ATMP directive that will enable a more patient-centred treatment approach within European legislation.

126

O-045 – A 3-Dimensional osteogenic-like structure from human autologous adipose mesenchymal stem cells: Reproducibility, Genetic stability, clinical safety/efficacy D. Dufrane

Endocrine Cell Therapy Unit/Centre of Tissue and Cell Therapy, University clinical hospital Saint-Luc,

Université catholique de Louvain, Brussels, Belgium.

INTRODUCTION. This work reports the potential of a 3-dimensional osteogenic autologous in term of human AMSCs differentiation in 3D,genetic stability and clinical safety/efficacy to cure a oncogenic/congenital large bone defect. METHODS. AMSCs isolation/differentiation into a 3Dimensional « bone-like » structure were performed:(i) Five patients with bone tumour characterized by several clonal cytogenetic alterations (study of tumor suppressor gene loci such as TP53/17p13,CDKN2/9p21,RB1/13q14) of the original tumour and (ii) Three patients with congenital pseudarthrosis. Graft characterization and genetic analysis (Karyotype/FISH) were performed on AMSCs proliferation/differentiation phases. Microarrays analysis studied the gene expression for osteogenic (RUNX2,BMP2,OPN3,FGF2,ALPL,SP7,FGF23,SMAD9,MEN1) and senescence/tumorogenic (c-Myc,TP53,NFKB,Cyclin D) development between un-/and osteogenic-differentiated states of AMSCs. The clinical safety/efficacy of the 3D was clinically followed biologically/radiologically post-transplantation. RESULTS. A mean of 65 ± 22 days (proliferation phase) was required to obtain a pure population of AMSCs in view to achieve the osteogenic 3-D (after 59 ± 17 days of differentiation). The microarrays analysis demonstrated the up-regulation of osteogenic genes for differentiated cells (p<0.05) and no sign of upregulation for TP53/NFKB/Cyclin D/c-Myc for both un-/differentiated AMSCs. In Group 1, no native tumour anomalies were found prior/after osteogenic differentiation of AMSCs. However, AMSCs culture can induced, in both Group 1 and 2, tri-/tetraploidies (0,5-14% of cells), recurrent clonal alterations as trisomy 7 (in 6-20% of cells for 3 patients) and chromosomal breakage cht(3)(q13.3) (for 4 patients) for undifferentiated AMSCs in proliferation phase. Interestingly, the osteogenic differentiation reduced significantly anomalies found in proliferation state (trisomy 7: <2-5.5% of cells). No sign of inflammatory reaction was found at <1 week and >1 month post-implantation. A total bone fusion was found after a mean of 12 months in all tumour and congenital pseudarthrosis contexts, respectively. CONCLUSION. These preliminary study demonstrated: (i) the reproducibility to obtain the 3D structure from all autologous AMSCs and (ii) the safety and efficacy of this clinical procedure.

127

O-046 – Development of a scaffold for human amniotic membrane handling and/or storage F. Gindraux,1,2,3 R. Laurent,2,4 M. Brennan,5 P. Layrolle,5 L. Nicod,2 L. Obert1,2 1 Orthopaedic and Traumatology Surgery Service, University Hospital of Besancon, France 2 Intervention, Innovation, Imagery, Engineering in Health (EA 4268), SFR FED 4234, University of Franche-

Comté, Besancon, France 3 Clinical Investigation Centre in Biotherapy, University Hospital of Besancon, France

4 Paediatric Surgery Service, University Hospital of Besancon, France

5 Inserm U957, Laboratory Physiopathology of Bone Resorption, Faculty of Medicine, University of Nantes,

France

Introduction: The human Amniotic Membrane (hAM) has good potential to help tissue regeneration thanks to its properties, i.e. as a membrane containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Goal: We aimed to use hAM as an Advanced Therapeutic Medicinal Product for bone repair in orthopaedic and maxillofacial surgery. To this end, we evaluated (1) the possibility to bond the allograft on a biocompatible scaffold to improve storage and/or allow handling during surgery; and (2) the effect of the scaffold on cell viability and phenotype. Methods: hAM from Caesarean delivery was provided by a local tissue bank. A biodegradable nanofiber jet sprayed polycaprolactone (PCL) scaffold (~500 μm thick) was produced using a novel jet spraying technique and provided by Biomedical Tissues (Nantes, France). hAM was cultured in contact with PCL scaffolds in two culture media used for mesenchymal stem cell (MSC) expansion or osteodifferentiation. These montages (hAM + PCL) were then grafted into an ectopic murine model (subcutaneous site) and explanted after 1, 2, 4 and 8 weeks. Cell viability and phenotype (and especially osteogenic potential) were evaluated in both in vitro and animal studies. Results: Currently, specifications of the PCL scaffolds are being reviewed and in particular, with a view to improving adhesion properties and thickness. Preliminary results showed that in in vitro

studies, PCL scaffolds maintained cell viability and osteodifferentiation. In animal studies, hAM and PCL scaffolds are biocompatible because no host reaction was observed against either the scaffold or hAM. Further results will be available at the time of the congress. Conclusion: The association of hAM allograft with a biocompatible scaffold will certainly improve handling during surgery. Since the scaffold maintains cell viability and osteodifferentiation, it can be grafted with the tissue on the bone defect. Thus, we purport that this scaffold may have other applications for tissue allografts requiring surgical handling.

128

O-047 – The use of high-definition optical coherence tomography and reflectance confocal microscopy to evaluate cellular and acellular human dermal matrices J.-P. Draye,1 M. A. L. M. Boone,2 G. Verween,1 A. Aiti,3 G. Verbeken,1 D. De Vos,1 T. Rose,1 S. Jennes,1 J.-P. Pirnay,1 G. Jemec,4 V. del Marmol2

1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology, Burn Wound Centre,

Queen Astrid Military Hospital, Brussels, Belgium 2 Department of Dermatology, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium

3 Emilia Romagna Regional Skin Bank and Cell Factory, Burn Center Bufalini Hospital, Cesena, Italy 4 Department of Dermatology, Roskilde Hospital, Health Sciences Faculty, University of Copenhagen, Denmark

Early excision of necrotic tissues and early skin grafting are essential for severely burned patients. For those patients with limited donor sites availability, accessibility to living skin equivalents (LSE) would be valuable. The purpose of this study was to evaluate the use of noninvasive assessment by High-Definition Optical Coherence Tomography (HD-OCT) and Reflectance Confocal Microscopy (RCM) to characterize the quality of both Decellularised Human Dermal Matrices (DHDMs) and cell-repopulated DHDMs in comparison with classical histology. Cryopreserved allogenic human skin (0.2-0.4 mm thick), obtained from deceased human donors after ethics committee approval, was used to prepare DHDMs. Four different decellularisation methods were used to prepare the DHDMs. To ease the removal of the epidermis, the allogenic skin samples were first incubated either in the presence of Dispase II (2.5 U/ml) at 4°C for 24h and at 37°C for 2h, or incubated in the presence of NaCl (1M) at 37°C for 24h. The dermal matrices were subsequently incubated either in 0.5% Triton X-100 or in 0.1% Sodium Dodecyl Sulfate (SDS) for 24h at 25°C with continuous agitation (75 rpm). NaCl treatment had small impact on collagen and elastic fibers of the DHDMs, and collagen type IV and VII of the epidermal basement membrane were immunodetected. By contrast, with Dispase treatment, the elastic fibers of the DHDMs were fragmented and collagen type IV and VII of the epidermal basement membrane were no longer immunodetected. Acellularity of the DHDMs was more easily achieved with SDS, but HD-OCT and RCM images showed that the three dimensional architecture of dermal papillae and vascular spaces were better preserved with Triton X-100. The optical techniques (HD-OCT and RCM) are useful to show that the methods used for the preparation of the DHDMs affect the quality of the DHDMs. For recellularisation of the DHDMs by different skin cell types, care must be taken in choosing appropriate processing steps to prepare the DHDMs in order to maintain particular properties of the dermal matrix for the development of LSE.

129

O-048 – Human mesenchymal stromal cells expanded in vitro in media supplemented with human platelet lysate M. Skific,1 M. Golemovic,1 S. Mazic,1 I. Bojanic,1 I. Humar,1 S. Davidovic Mrsic,2 K. Crkvenac Gornik,2 I. Ilic,3 N. Durakovic,4 M. Mikulic,4 R. Serventi-Seiwerth,4 B. Labar,4 B. Golubic Cepulic1

1 Department of Transfusion Medicine and Transplantation Biology, Zagreb University School of Medicine and Clinical Hospital Centre Zagreb, Croatia 2 Department of Pediatrics, Zagreb University School of Medicine and Clinical Hospital Centre Zagreb, Croatia 3 Department of Pathology and Cytology, Zagreb University School of Medicine and Clinical Hospital

Centre Zagreb, Croatia 4 Division of Hematology, Zagreb University School of Medicine and Clinical Hospital Centre Zagreb,

Croatia

Introduction: Human mesenchymal stromal cells (MSCs) are nonhematopoietic multipotent cells that express regenerative and immunomodulatory properties in vitro and appear immunoprivileged what gives them considerable therapeutic potential. Goal: The aim of this study was to evaluate biological properties of MSCs expanded in vitro in the presence of human platelet lysate (PL). Methods: Mononuclear cells (MNC) were isolated from the bone marrow of 13 healthy individuals by Ficoll density gradient centrifugation and plated at 160000 MNC/cm2 in media supplemented with 10%FBS (fetal bovine serum), 10%PL or 5%PL. Upon reaching 80% confluence, cells were harvested by trypsinization and replated at 1000 MSCs/cm2 until reaching passage 4 (P4). The growth kinetics of MSCs cultured in different culture conditions was evaluated by light microscopy. Clonogenic potential of MSCs was evaluated by colony forming unit-fibroblast (CFU-F) assays performed at each P. Whenever sufficient number of cells was obtained, further tests were performed. Immunophenotype was assesed by flow cytometry. Multipotency was evaluated by inducing MSCs into adipogenesis and osteogenesis in vitro. Immunomodulatory properties were evaluated by adding MSCs to mixed lymphocyte reaction (MLR) (n=1). Karyotype analysis was performed on MSCs harvested in P2 or P3. Results: Cell growth analysis showed dominant growth of MSCs in the presence of 10%PL which was also confirmed by CFU-F tests. The median time needed for one MSC population doubling in the presence of 10%FBS was 144hr compared to 46hr and 36hr in 5%PL and 10%PL, respectively. More than 96% of cells cultured in the presence of 10%PL expressed CD90, CD73 and CD105 in P1. Cells cultured in the presence of 10%PL differentiated into adipocytes and osteoblasts. In MLR experiment, MSCs cultured with 10%PL displayed stronger immunomodulatory effect compared to those cultured in 5%PL. Karyotype was analysed in 15 cell samples obtained from different donors and culture conditions (5% and 10%PL). Analysed metaphases displayed abnormal karyotype in three samples; all of them originating from MSCs cultured in the presence of 5%PL. Conclusion: MSCs cultured in the presence of 10%PL display expected biological and immunomodulatory properties. This study provides a basis for further evaluation of PL as a valuable FBS substitute in clinical grade MSCs expansion.

130

O-049 – Development of ATMPs from bench to patient: how to navigate the European regulatory framework P. Celis

European Medicines Agency

In 2009, legislation came into operation for Advanced Therapy Medicinal Products (ATMPs), which are defined as Gene therapy and Somatic Cell therapy Medicinal Products and Tissue Engineered Products. This legislation (Regulation (EC) No 1394/2007), apart from setting a clear legal framework and regulatory pathway for these innovative medicines, also established a new Scientific committee at the European Medicines agency (EMA), the Committee for Advanced Therapies (CAT). In my talk I will address;

− The regulatory framework leading to a marketing authorisation of an ATMP

− What a ATMP developers should take into consideration already in early development phases to facilitate future regulatory submissions (e.g. for clinical trials, for marketing authorisation).

− How to make best use of the regulatory procedures at EMA: ATMP classification, ATMP certification and Scientific Advice for ATMPs.

− Further Initiatives in place at EMA/CAT to assist companies developing ATMPs. This includes initiatives set-up in the framework of the CAT Workprogramma 2010-20151, the increased possibility for interactions with the EMA in early development (meetings with the EMA Innovation Task Force and the CAT) and the assistance offered by the EMA SME office2.

1 See: http://www.ema.europe.eu/docs/en_GB/document_library/Work_programma/2010/11/WC500099029.pdf

2 See:

http://www.ema.europe.eu/ema/index.jsp?curl=pages/regulation/general_content_000059.jsp&mid=WC0b01ac058

00240cc

131

O-050 – Antibiotic decontamination of banked tissues - a microbiologist's viewpoint T. L. Pitt, C. P. McDonald, R. Lomas National Transfusion Microbiology Laboratory, NHS Blood and Transplant, England

Tissues retrieved from deceased donors are frequently contaminated with bacteria which originate from the endogenous flora of the donor or exogenously from retrieval staff and environment. These bacteria may represent a potential hazard to recipients of the tissue. Non viable allografts can be effectively sterilized by gamma irradiation but cryopreserved viable allografts need to be decontaminated by other means. Several antimicrobial cocktail solutions of differing composition and concentrations are widely used to decontaminate viable banked tissue allografts at different temperatures and times of exposure. A recent survey by de By et al. (2012; HSR Proc Intens Care Cardiovasc Anesth;4:251-260) of decontamination methods in 17 European cardiovascular tissue banks showed that each used a unique formulation selected from 25 antibacterial and antifungal compounds. The cocktails ranged from mixtures of 3 to 6 agents with broad or narrow spectra of activity. Most banks incubated tissues in antibiotic solutions at 4°C and the duration of exposure ranged from 6 to 72 hours. We compared the efficiency of 4 cocktails comprising nine antimicrobials to kill suspensions of 27 strains of 13 bacterial species, and 3 Candida spp. at 4°C, 22°C and 37°C for 24 hours. All, but one, bacterial strains were susceptible to one or more of the agents tested individually at concentrations of at least 4-fold below the recommended susceptibility breakpoint concentrations for drug/species combinations. The concentrations of several of the cocktail constituents were often greatly in excess (50-1000 fold) of that required to inhibit the growth of susceptible strains. Each cocktail was most efficient at 37°C, less so at 22°C, and poorly active at 4°C. The practice of decontamination of tissues with antimicrobials at low temperatures is therefore not supported by in vitro susceptibility tests. A further study of 67 isolates of over 30 bacterial species recovered from ‘failed’ post decontaminated heart valves showed that the great majority were indeed susceptible to the antibiotic cocktail used. These findings suggest that antimicrobial resistance is not the key reason for decontamination failures but the delivery of optimal cidal concentrations of agents and their penetration into the tissue at a temperature appropriate to their antibacterial mode of action may be of more relevance.

132

O-051 – Emerging infectious disease outbreaks and the safety of tissues and cells transplantation in Europe D. Domanović

European Centre for Disease Prevention and Control, Stockholm, Sweden

Transmission of infectious diseases is infrequent but serious adverse effect of tissues and cells transplantation. Recent outbreaks of several emerging infectious diseases (EIDs) in Europe, resulting from ecological, environmental or demographic changes, pose a threat to the transplantation safety and the sustainability of tissue and cell supply. This presentation provides an update on these outbreaks, the attributable risks, and strategies to prevent and control EID transmission through tissues and cells. Transplanted patients can acquire EIDs through infected cells and tissues, blood and blood components, or through community exposure. Emerging infections are unexpected in recipients and oftentimes unrecognized in donors due to low availability and performance of donor evaluation procedures. This increased risk is amplified by immunosuppressive therapy, specific allograft factors and extensive exposure to the hospital environment which all predispose transplant recipients for such infections. During EID outbreaks supply with tissues and cells could also be adversely affected because of the donor shortages and resource problems. Europe witnessed the first outbreak of Chikungunya fever outside the tropics in 2007 with some 250 cases in Italy, transmitted by the invasive Asian tiger mosquito. During the summer heat wave of 2010, a WNF outbreak erupted in new areas of Southeast Europe; significant temperature deviations over a 30 year average were associated with an unprecedented upsurge, involving over 900 confirmed cases. Since 2009 Greece has experienced a resurgence of indigenous cases of malaria transmission caused by Plasmodium vivax and Madeira is experiencing the first sustained transmission of Dengue fever in ‘Europe’ since 1920, with over 2,000 cases as of December 2012. These recent outbreaks have provided important lessons on how transplant patients are affected, and how transplant programmes should be managed in the face of emerging infections. Responses to these threats posed by EIDs include assessing the risk to the recipient’s health and supply, evaluating how best to prevent, manage and/or control each potential risk and communication this information to donors, recipients, physicians and the general public. Specific activities undertaken in EU to mitigate the EID threats entail diseases surveillance, prioritizations, risk assessments, mapping of outbreaks, preparedness plans and research.

133

O-052 – Towards a common microbiological testing protocol of human tissues and cells for transplantation? V. Saegeman

Department of Laboratory Medicine and the Department of Hospital Epidemiology and Infection Control of University Hospitals Leuven, Belgium

The European regulation (2004/23/EC and 2006/17/EC) requires human tissues and cells to be safe for the recipient. This safety must be documented and every step in their production must be validated. This also applies to microbiological safety. However, specific microbiological tests required are not prescribed in these regulations. Indeed, much difference exists in the processing of the banked human tissues and cells: e.g. skin, fetal membranes, musculoskeletal, tympano-ossicular and ocular allografts but also reproductive cells, cell cultures, hematopoietic stem cells and cord blood. Likewise, there is a large variety in microbiological techniques (which and number/volume of samples, timing of sampling, incubation conditions and culture media,…) to test for the absence of microorganisms on these products. In order to overcome the existing heterogeneity in microbiological techniques among different cell and tissue banks, a consensus is presented based on the European Pharmacopoeia chapter of sterility. Obviously, one has to take into account the paucity of material available for additional biological tests in this setting and the final application of the different tissues and cells. The latter leads to different criteria for acceptance of the different products for transplantation

134

O-053 – Optimalization of bone/tendon tissue procurements in term of microbiological contamination V. Vandenschrik, T. Schubert, M. van Steenberghe, L. Lekeux, X. Merny, D. Dufrane UTTCAL - Cliniques universitaires Saint-Luc, Brussels, Belgium

Introduction: The contamination of bone/tendon allografts remains the major hurdle of a widespread use of human tissues in clinical practice. Therefore, this work aims to study the impact of procurement procedures on the rate of tissue contamination Material/Methods: Among 284 human tissue procurements (2009-2013), corresponding to 6637 procured bones/tendons, 3 experimental groups were designed: (i) procurements performed in the autopsy room (30 donors-353 grafts) in comparison to tissue procurements performed in the operating theatre with (ii) non- sterilized microbiology vials (97 donors-2488 grafts) and (iii) sterilized vials (157 donors-3798 grafts). The number of contaminated tissues, procurement time and the rate of contaminated tissues (with the germ origin) were collected. The microbiological testing of the mortuary room was investigated in term of air contamination in view to correlate with the contamination of procured tissues. Each procurement conditions were statistically compared. The impact of time procurement on the graft contamination was also studied following a clustering method: 2h30 (60 donors/984 grafts), 4h (70 donors/1977grafts) and 5h30 (23 donors/775 grafts). Results: No correlation was found between the microbiological contamination of the mortuary air and the rate/origin of grafts contamination. A significant reduction by 53% of contaminated graft was found for tissue procurements performed in operating theatre in comparison to autopsy room (9.6 vs. 4.5% of contaminated grafts, p<0.001). Secondly, a significant decrease by 18% of contaminated tissues was also demonstrated when microbiological samples were collected in sterilized vials in comparison to non-sterilized vials (3.7 vs. 4.5%, p<0.005). Tissues procurement in operating room with a procedure of sterile vials for microbiological sampling decreased by 25% the number of excluded grafts (p<0.05). Although the number of harvested tissues was correlated to the time of procurement 16 ± 6 vs. 28 ± 9 vs. 34 ± 8 of procured tissues for 2h30 vs. 4h vs. 5h30,respectively, p<0.05), no impact of the procurement time on the rate of grafts contamination was found. Conclusion: This study demonstrated that the rate of contaminated human tissues can be minimized following a procedure in a operating theatre with sterile processing of microbiological sampling. In addition, the contamination rate of procured tissues is not impacted by the time of procurement when the procedure is performed in controlled conditions.

135

O-054 – False negative results in microbiological analyses of corneal tissues R. Mistò,1 A. Pocobelli,2 F. Pateri,1 A. Limongelli,1 L. Giurgola,3 C. Gatto,3 J. D'Amato Tothova3 1 Monza Eye Bank, Monza, Italy

2 Rome Eye Bank, Rome, Italy 3 Al.Chi.Mi.A. S.r.l., Ponte San Nicolò, Italy

Introduction: The use of antibiotic cocktails during corneal processing can lead to an antibiotic carry-over effect, which in turn can generate false negative results in microbiological analysis. Purpose: The aim of the study was to investigate the impact of antibiotic residues on microbiological analyses of organ cultured donor corneas. Material and methods: Sixty-one corneal tissues unsuitable for transplantation were retrieved by the personnel of two Italian eye banks and transported to the bank in Eusol-C (Al.Chi.Mi.A. S.r.l, Italy); once transferred to Tissue-C (Al.Chi.Mi.A. S.r.l, Italy) corneas were stored for 12-14 days at 31°C. Microbiological analyses were performed post-processing on Tissue-C by the eye banks, according to bank standard procedures using BacTEC plus aerobic/anaerobic system (BD); in parallel, microbiological analyses were performed by Al.Chi.Mi.A. S.r.l. with sterility test according to the European Pharmacopeia after removing potential interfering antibiotics with ResEP™ device (Al.Chi.Mi.A. S.r.l, Italy). Results: Sterility test after removing potential interfering antibiotics with ResEP™ device showed in average 59% of positive (contaminated) samples at the end of the process; the percentage of positive results varied depending on the Bank. None of these contaminations was detected by BacTEC. Conclusions: Removal of antibiotic residues from corneal storage media with the ResEP™ device resulted in a significant number of false negative results in the microbiological analyses. The presence of contaminants in the media at the end of the storage process indicates that standard corneal storage media doesn’t guarantee efficient corneas decontamination. Authors L. G., C. G. and J. D. are employed in the R&D department of Al.Chi.Mi.A. S.r.l., the Company that manufactures the products discussed in the present abstract.

136

O-055 – Bacteriology testing of cardiovascular tissues: comparison of transport solution versus tissue testing in two different labs R. Díaz Rodríguez, B. Van Hoeck, B. Mujaj, R. Ngakam, Y. Fan, R. Jashari European Homograft Bank, International Association, Brussels, Belgium

Introduction: Bacteriology testing is mandatory for quality control of recovered cardiovascular allografts (CVA). Testing of transport solution was recommended for many years as the initial evaluation of tissue sterility. Due to different regulations in different EU member states, additional direct testing of the tissue fragments was recommended. Aim: To compare the efficiency of two different methods in germ detection, either in the transport solution or directly in the tissue fragments of recovered tissues. Material and Methods: Routinely the recovered CVA are evaluated for contamination in three steps: A-transport solution, B-pieces of tissues after incubation in antibiotics, and C-cryopreservation solution. For A test, 50 ml of the transport solution was filtered through 0.45 µm cellulose membrane and tested in one laboratory (A1) whereas the CV tissues (myocardium, mitral and tricuspid valves, the aortic and pulmonary wall, arteries) in a second laboratory (A2). Solutions and tissue samples were incubated in thioglycolate medium for aerobe/anaerobes at 30-35 °C and in soya-bean casein enriched medium at 20-25 °C for fungi and yeasts. Samples are incubated during 14 days with a daily control of turbidity. Germ identification is done in all A positive samples. This study included 274 donors of CVA (hearts/arteries). Results: Transport solution (A1) tested positive in 42.1 % of hearts and in 50.5 % of arteries, whereas the tissue samples (A2) tested positive in 32.4 % of hearts and 37.6 % of arteries. The main species identified in both A1 and A2 were Staphylococcus spp. in 55 and 26 % of cases, and Propionibacterium spp. in 8 and 19 %, respectively. There were mismatches between A1 and A2 in 27.6 % of total tests. 67.4 % of mismatches are identified as positive A1 whilst 32.6 % positive A2. The final testing (B/C) showed that 71.6 % of positive samples in test A1 became sterile after treatment with antibiotic cocktail whereas 64.8 % of positive A2 cases became sterile after decontamination treatment. Conclusion: Bacteriology testing of transport solution showed more positive cases than direct incubation of the tissues. Furthermore, the final decontamination of the tissues was better in the culture positive cases from the group A1. Nevertheless, due to the mismatches and different bacteria identification with those two methods, testing of both the tissues and the transport solution might give significantly better detection of tissue contamination at the beginning of processing.

137

O-056 – Tissue Vigilance: Lessons learned from the SOHO V&S Project D. Fehily

Italian National Transplant Centre (CNT)

An effective vigilance and surveillance system plays a pivotal role in enhancing the safety of tissue and cells for human application. In some cases it facilitates rapid intervention by professionals or regulators to prevent further harm when an adverse outcome has been detected. In general, it ensures the sharing of invaluable information to support improvements in systems and procedures for the benefit of donors and patients. The SOHO V&S Project (Vigilance and Surveillance of Substances of Human Origin) was a three-year, EU-funded project led by CNT that finished in February 2013. It provided an unprecedented opportunity for Competent Authority vigilance officers and relevant professionals to explore, in-depth, how best to organise vigilance systems for tissues and cells in the EU. The key objective was to develop a shared view of how serious adverse events and reactions associated with tissue and cell donation or human application should be reported, evaluated, investigated and communicated within the EU. Thorough and effective detection and investigation of adverse events and reactions are critical to ensuring that the lessons emerging from each adverse incident are used for preventing recurrence, whenever possible. Guidance developed in the project highlights the role of the clinical user in suspecting that the tissues or cells might be the cause of unexpected clinical symptoms. Once SAR/E have been detected and notified, investigation becomes the priority. The project published guidance on the appropriate approaches to confirming imputability for infectious and malignant transmissions and for conducting root cause analysis for SAE. The working groups drew on the outcomes of the NOTIFY project, an initiative led by the World Health Organization, in which SOHO V&S participated, where experts from across the globe gathered information on documented cases of adverse reactions and events for organs, tissues, cells, gametes and embryos and made them publicly accessible on a searchable database (www.notifylibrary.org). To ensure effective dissemination and application of the guidance principles developed in the SOHO V&S project, training was delivered, with a combination of e-learning followed by a residential module, for vigilance officers from EU Competent Authorities.

138

O-057 – Model of Cooperation between National Competent Authority (NCA’s) and Police

F. Teskrat

Combating trafficking in human organs and tissues, however, is challenging. In a 1998 report on human tissue transplantation crime, 1 its authors stated that ‘illegality arising from the transplantation of human tissue exists primarily because of an international shortage of donors able to provide tissue suitable for use in recipients’. A report from the United Nations Economic and Social Council2 indicated that ‘some countries do not have the resources and capacity to respond adequately to the problem…, because awareness, law enforcement resources, judicial expertise and cooperation between national and international law enforcement agencies are insufficient or lacking. Effective law enforcement efforts and international cooperation are necessary’. In the field of organ transplantation, it is acknowledged that illegal activity is focused on trafficking. In tissue and cell donation, banking and transplantation, however, there are recognised IFA (Illegal and Fraudulent Activities) that go beyond trafficking to include issues such as false advertising, fraudulent authorisation and illegal importation. Biomedical innovation has led, in recent years, to an upsurge in the use of human tissues and cells, as well as cellular or tissue-based products, to such an extent that their use has been referred to as an ‘industry’. Today, these human substances, ranging from musculoskeletal, cardiovascular and ocular tissues, to cord blood and many types of stem cells, are used routinely for medical purposes, therapy and research. This burgeoning field, which involves both private (for profit and not-for-profit) and public sector tissue establishments is, in some cases, highly lucrative. Thus profit may be the motivating factor for the provision of tissues and cells. In such instances, the risk that an illegal or fraudulent activity (IFA) might occur must be acknowledged and, indeed, a number of cases have been well documented. Requirements for ensuring the quality and safety of tissues and cells in the EU are set out in Directive 2004/23/EC3 and its implementing measures (Commission Directives 2006/17/EC4 and 2006/86/EC5). Although no specific reference to IFA is made, Article 27 of Directive 2004/23/EC does require EU Member States, when transposing the Directives into their national legislation, to establish rules for the penalties to be applied when violations occur and to ensure that these are enforced. There is, therefore, an obligation upon NCAs in the EU, and those designated by them to conduct inspections, to be aware of issues related to IFA and to be on the alert. ANSM carried out inspections performed on the basis of an established programme focused on the suspicion of IFA. It is a strategy that responds to circulation of cells and tissues products defined by European Directive. One of the priorities for the inspectors, based on the increasing

1 No.87 Human tissue transplantation crime. Elizabeth King and Russell G. Smith. B. Trends & issues in crime and criminal justice. May 1998. 2 Preventing, combating and punishing trafficking in human organs. Report of the Secretary-General: United Nations Economic and Social Council. 21 February 2006. CN.15/2006/10. 3 Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004. Official Journal of the European Union. L102, 7.4.2004. p.48 4 Commission Directive 2006/17/EC implementing Directive 2004/23/EC of the European Parliament and of the Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells. Official Journal of the European Union. L38, 9.2.2006. p.40 5 Commission Directive 2006/86/EC implementing Directive 2004/23/EC of the European Parliament and of the Council as regards traceability requirements, notification of serious adverse reactions and events and certain technical requirements for the coding, processing, preservation, storage and distribution of human tissues and cells. Official Journal of the European Union. L294, 25.10.2006. p.32

139

number of illegal activities, involves, at least, the control of ethical aspects related to consents given by donors close relatives and local procurement conditions. In 2012, ANSM in cooperation with the Police had carried out inspection in a tissue bank who wanted to distribute capsule of human placenta. These products (claimed by private companies to have medicinal properties) are not regulated in the same way all over EU countries. The topic that will be presented during the EATB congress, will be the results of this cooperation.

140

O-058 – Identificaton errors and double check procedures M. J. Happel, M. S. E. Bergers, P. Y. Zijlker-Jansen, J. C. Wiersum-Osselton, A. G. B. Bokhorst

TRIP Dutch National Hemovigilance and Biovigilance Office

Objectives: Since August 2006 TRIP is collecting reports of serious adverse reactions and events (SARE) which may be related to quality or safety of human tissues or cells on behalf of the Competent Authority. Patient safety aspects including accurate identification of donor and patient and correct selection of tissues and cells during processing and distribution is an important area of biovigilance. This abstract gives an overview of the reported events of identification and product selection errors from 2006 to 2012. Methods: Reports are submitted to TRIP online or by paper form and categorised based on definitions which are available on the TRIP website. These reports are initially assessed by TRIP staff; further information is requested if necessary. A panel of expert professionals reviews all reports. TRIP publishes an annual report with aggregate information and recommendations for improvement of safety. Results: In 2006-2012 484 reports of adverse reactions and events were received. Of this total, 36 reports concerned an identification error which resulted in transplantation or transfer of incorrect or inappropriate tissues or cells, loss of cells or tissues, near miss or minor consequences. The reports involved gametes and embryos, skin, chondrocytes, peripheral blood stem cells, cornea and tendon. Identity checks were performed by one or two persons. Two persons could be either two professionals or one professional and the patient or the patient’s partner. In six reports it was not stated how many persons were involved. Table 1 shows the reports of identification and product selection errors in relation to the persons involved and the outcome. Table 1. Reports of identification and product selection errors. Persons

performing

check

Incorrect

tissues or cells

transplanted

Loss of tissues

or cells

Near miss

Minor

consequences

Total number

of reports

2 professionals 2 3 6 2 13

1 professional

and

patient/partner

1 2 3 0 6

1 professional 4 6 0 1 11

Not stated 1 2 1 2 6

Total 8 13 10 5 36

Conclusions: The worst outcome of an identification error is the transplantation or transfer of incorrect tissues or cells or the loss of tissues or cells. The majority of these outcomes occurred when identification or selection was performed by only one person. When checks were performed by two professionals, the majority of cases resulted in near miss. The double check system is essential in the chain of tissue and cell transplantation to prevent misidentification and product selection errors and adverse outcomes.

141

O-059 – Clinical strategy of application of deep frozen-radiation sterilised bone allografts W. Marczyński, J. Białecki, M. Obrębski, A. Kolbuszewski Post Graduate Medical Education Centre in Warsaw, Teaching Othopedic Hospital in Otwock, Poland

Orthopaedic and posttraumatic damage of osseous tissue usually requires surgery. Often the quantity of osseous tissue is inadequate to ensure reconstruction. Lack of local osseous tissues reserves calls for the use of auto- or allogenic bone grafts. We have successfully used allogenic, biostatic deep frozen bone grafts sterilized by radiation. Strategy for the use of grafts is varied. The strategy arise from the biology and biomechanics of the implanted grafts for various indications. Orthopaedic indications include: primary and revision arthroplasty of the hip, including realoplasty post inflammatory, osteotomy of the tibia, benign tumours. Indications traumatic application grafts related bone loss, impaired non-union and reconstructive surgery. We used pieces of spongy bone in the form of morrselised bone grafts. The biological role of grafts related to supplement use of bone defects and for healing and graft remodelling. Biomechanical role is to prepare the technical specifications for the foundation of hip replacement prosthesis load transfer for a transplant. Remodelling bone grafts were evaluated on the basis of physical and radiological. The results were evaluated from 2 to 5 years after surgery. The study was conducted at regular intervals. We will present examples of the use grafts in specific clinical situations. Conclusions: 1. The use of allografts, frozen biostatic allows you to limit the scope and duration of the

operation. 2. Allogeneic frozen, biostatic radiation sterilized bone grafts are reconstructed by "creeping

substitution" in 3 to 6 months after surgery. 3. The progress of graft remodelling depend on its structure and the site of

implantation.

142

O-060 – Endothelial cell transplantation on synthetic vascular bypasses J. Meinhart, N. Hownietz, A. Stümpflen, M. Gorlitzer, M. Grabenwöger Department for Cardiovascular Surgery, Hietzing Hospital, Vienna, Austria

Introduction and Purpose: We report on the long term clinical use of a ATMP product. Synthetic vascular bypasses for vascular reconstructions in patients with severe atherosclerosis have a poor long term prognosis, Occlusions of such bypasses often lead to major amputation and death. The main reason for this phenomenon is a direct contact of the blood with the synthetic material due to a lacking endothelium. We developed a method to cover the synthetic bypasses with cultured autologous endothelial cells, thus reconstructing the natural barrier between blood and the rest of the body. Patients and Methods: In a first operation a short segment of a subcutaneous vein was excised and, autologous endothelial cells were harvested and cell cultures established.. Cells were cultured until enough cells were available for the confluent lining of the inner surface of synthetic bypasses. Every graft was individually matched with the anatomical needs of a given patient. The endothelium covered bypass was then implanted in second operation. Patients were examined regularly in a dense follow up scheme. Results: 370 endothelialized bypasses have been implanted in different anatomical positions. No method related complicatens occurred, especially there were no bypass infections or neoplasms. Occlusion, amputation and mortality was significantly reduced when compared to untreated bypasses. The overall primary patency rate of endothelialized bypasses was 69% at 5 years and 61% at 10 years. Seven grafts had to be explanted due to surgical reasons. These grafts could be examined morphologically. In all cases a confluent endothelium was present. In some areas atherosclerotic changes were visible at various degrees. In all grafts, signs of vessel wall formation could be detected, proving full integration of the bypasses into the body. Conclusion: Our program for autologous endothelial cell transplantation in vascular surgery was started 1989. This makes it the second oldest advanced therapy medicinal product (ATMP) in continuous clinical use in the world, only surpassed by the transplantation of autologous keratinocytes for burned skin. We could prove that the therapeutic use of cultured autologous cells is a save and clinically feasible method. Endothelial cell transplantation significantly reduced occlusion of bypasses and the need for reoperations. Furthermore, amputation and thus mortality rate was also reduced significantly.

143

O-061 – Outcome of corneal transplantation D. Ponzin

The Veneto Eye Bank Foundation, Venice, Italy

The cornea is the most transplanted tissue and penetrating keratoplasty (PK) the procedure most largely applied. Advances in corneal surgery have led to the development and increasing numbers of lamellar keratoplasty (LK), as a way to replace only the anterior stroma or the posterior stromal/endothelial layers. When seeking outcomes of corneal transplantation and risks affecting graft survival, it is essential to consider that multiple factors can potentially have an effect (corneal processing, patient demographics, graft indications, surgery, postoperative treatment, and the clinical course) and that all these factors could widely change among different groups of patients and postoperative graft management strategies. Health-related quality of life and patients’ satisfaction are further indicators to be investigated. In our experience, corneal ectasia remains the most common indication (49%), followed by regraft (16%) and pseudophakic corneal edema (PCE, 9%). The overall, 5-years rate of graft failure is 11%. Adverse reactions and complications (other than early and late graft failure) are reported in 3% of patients in the first postoperative week and in 23% during a 5 year follow-up period. The probability of 5-year survival is 83%, best in eyes with ectasia/thinning (96%) and less favorable in PCE (67%) and regraft (64.%). Multivariate analyses showed the following variables to be linked to an increased risk of graft failure: regraft for any reason, all clinical indication except PCE, history of ocular inflammation/infection, pseudophakic/aphakic eye, vitrectomy, adverse reactions/complications, and surgeons’ low caseload. Grafting improves health-related quality of life results of patients, influencing mental health (i.e psychological attitude, social interaction, emotions) with minor effects on physical health (limitation, pain, vitality). Endothelial Keratoplasty performed with Descemet's Stripping Automated Endothelial Keratoplasty technique exhibits visual outcomes, graft survival and complications (events and reactions) similar to those achieved after PK. The 100,000 or more corneal transplants carried out worldwide every year rely on the activity of eye banks. The cooperation between corneal surgeons and their source eye banks is a key factor for the permanent evaluation of benefits and risks of corneal transplantation.

144

O-062 – Evaluation of human amniotic membrane associated with dermal substitute in the management of full thickness burns A.-S. Hatzfeld,1 L. Pasquesoone,2 P. Guerreschi,2 A. Qassemyar,2 I. Martinache,1 P.-M. Danze,1 P. Marchetti1 1 Tissue Bank of Lille, CHRU Lille, Lille, France 2 Plastic Surgery Department, CHRU Lille, France

Third-degree burn patients benefit from treatment inducing full-thickness skin wound healing. At this step, dermal reconstruction is fundamental for improving function and aesthetic outcome. In this context, dermal substitutes are an appropriate way to minimize scar contraction and to optimize the quality of the grafted area. Besides it has been shown that amniotic membrane (AM) graft decreases the pain, accelerates the healing process, and reduces scar formation. Here, we have investigated the effect of AM associated with the Matriderm®, a dermal substitute, in the management of full thickness burns in a pig model. Four skin deep burns were performed in the back. One week after, dead tissue was removed. Then, area was specifically treated: Matriderm® alone, AM alone, Matriderm® + AM, or nothing as following: Matriderm® was applied on appropriate wound bed in. Then, autologous thin-thickness skin fragments were applied on four wounds. AM was put on the both appropriate wounds. At day 5, 10 and 31, biopsies were performed. AM has a benefic effect to diminish scar retraction. In the presence of AM, skin wound healing displayed a higher elasticity compared to the condition without AM. At day 31, histological examination showed elastic fibers constituting a dense network with long fibers with Matriderm® and AM. This network was similar to the healthy skin network. A similar network was detected with Matriderm® or AM alone. The presence of AM increases the early recruitment of fibroblasts leading to a fibroblast level similar to healthy dermis. Neovascularisation is recovered in dermis with Matriderm® and AM. Encouraged by these results, the combination Matriderm® + AM was tested in the context of a full-thickness skin wound healing in one human patient. The healing was full and fast. The dermis was well-integrated without any edema and inflammation. No infection wad detected. No early hand retraction was noticed. Altogether encourages us to consider the combination AM + Matriderm® as a good treatment in the context of deep burn care, especially with good clinical results on elasticity recovery. This study allows us to envisage its use especially in regions which need good elasticity and pliability.

145

O-063 – Acellular dermal matrix M. L. Pérez, E. Agustí, A. Savio, E. M. Martinez N. Otero, A. Villarrodona, E. Trias Transplant Services Foundation (T S F), Barcelona, Spain

The present study describes the development of a decellularization treatment to be applied on human skin from cadaveric donor where cytoplasmic and nuclear components are removed and basement membrane remains intact. The final product been a matrix derived from human dermis that lacks of immunogenic markers assuring minimum inflammation risk associated to implantation, avoiding even graft rejection. The purpose of the matrix is to be used in reconstructive surgeries for soft tissue augmentation able to be repopulated by recipient cells but it has been used also in various periodontal procedures, like root coverage procedures. Skin grafts with different characteristics were tested. Three different protocols based on chemical treatments were assayed in which salt solutions alone or together with different surfactant agents were compared. Histological assessments were performed in order to evaluate the absence of cells and the maintenance of the extracellular matrix structure. Residual toxicity test were carried out by chromatography assuring the safety of the matrix Cryopreserved skin was easily de-epithelized by stripping compared to glycerolized skin although histological results show equally absence of cells. Every protocol assayed assured cell removal. Tensile tests were performed to measure the effect of the decellularization method chosen on the mechanical properties of the tissue. Maximum load, maximum stress, and elastic modulus were assayed in a tensile tester. Deformation caused by the load was more pronounced for native samples than for decellularized ones. Parameters related to the thickness of the sample show the importance of thickness in resistance to breaking regardless to decellularization treatment. These results allow the bank the development of an acellular dermal matrix in different sizes and two different preservations for soft tissue restoring, successfully used in two maxillofacial surgeries.

146

O-064 – Are CO2 cleaned allogenic bone grafts (eCOO-technology) as a high local dose antibiotic carrier system (OSTEOmycin) a safe and sufficient option for treatment of implant related infections and chronic osteomyelitis in a one-stage revision procedure? K. Kaudela,1 W. Kaltenbrunner,2 M. Weissinger,2 B. Bader,2 C. Schenner2 1 European Cell and Tissue Bank (ECTB), Wels, Austria 2 LK Zwettl – Gmünd - Waidhofen Orthopaedic Department

Background: Our hypothesis is that a high local antibiotic load, which is also effective against biofilms, using CO2 cleaned allogenic bone grafts (eCOO-technology) as carrier, improves the efficacy of a one stage exchange and offers all advantages regarding patient comfort and economic. Therefore, we have taken the approach of potentially permanent metal spacers using antibiotic loaded CO2 purified allogenic bone grafts in a one-stage procedure. In this retrospective study we report our experience. Patients and methods: In the period from February 2008 to March 2013 in our department 18 one-stage revision procedures (11 hips, 7 knees), 3 re implantations and 5 patients with chronic osteomyelitis have been revised. Previous exchange operations in the implant group: 1 two-stage knee, 1 single-stage knee and 1 patient with 1 single-stage and 1two-stage at the hip, and a total of 13 soft tissue revisions. Four patients had in addition 62 times previous VAC exchange at the hip (between 3 and 39 times per patient). In osteomyelitis group 1 patient was 8 times revised previously. All together 62 previous operations took place on 26 patients. In the implant group all patients underwent a removal of implant or spacer, accurate debridement and jet lavage was performed in both groups. Average operation time was 3.5 hours, hospitalization was 1 days, ASA score on average 2.6 in knee group, 2.7 in hip and 1.6 in osteomyelitis group. Results: There were no surgical related complications; no patient needed postoperative intensive care. Average follow-up was 22.4 months (knee), 19 m (hip), 24 m (replant) and 32.6 m (osteomyelitis group). One patient showed a cup loosening after a heavy crash on the revised hip 4 months after the revision. To date, all patients are free of infection. Conclusion: The use of CO2 cleaned allogenic bone grafts as a high dose antibiotic carrier system is a save and sufficient treatment option in a one stage revision procedure, as well as in the treatment of chronic osteomyelitis, and it improves the outcome compared to the so called ‘gold standard’ of a two stage procedure, offering much more patient live quality and enormous cost reduction. Author K. K. is the Responsible Person of ECTB.

147

O-065 – Treatment of bone non-union by a composite graft made of concentrated bone marrow and demineralized bone matrix processed in a cleanroom S. Cuppens,1 W. André,1 N. Aouassar,1 O. Cornu,2 D. Dufrane1 1 Unité de Thérapie Tissulaire et Cellulaire de l'Appareil Locomoteur, Cliniques Universitaires Saint-Luc,

Brussels, Belgium 2 Orthopedic Surgery Service, Cliniques Universitaires Saint-Luc, Brussels, Belgium

Introduction: Bone non-union remains a major health problem in orthopaedic surgery due to co-morbidity factors (Type 2 diabetes, smoking,...). Goal: This study assessed the potential of “a composite graft made of concentrated bone marrow (BMC) supplemented with demineralized bone matrix (DBM)” processed in a cleanroom. Materials and methods: A mean of 164±48ml (72 patients, 20-88yrs) of non-concentrated bone marrow (BMN, collected during the surgery of bone non-union within a mean of 15±9min) was directly transferred to the cleanroom (6±4min) to be filtered/ concentrated by the SEPAX© (20±3min). The BMC was finally mixed with DBM and then immediately retransferred to the operative room for implantation at the bone non-union site. The total process in the cleanroom lasted an average of 55±12min. The relation between donor’s age/bone collection procedure/preparation time of the graft and bone marrow concentration on the BMC outcome (CD34, CD45, CFU as colony forming unit and cellular viability) was then assessed. Forty-six patients with a minimum follow-up at 1 year post-consolidation were radiologically investigated (classified as 0=non-consolidation/1=partial/2=total consolidation) for bone reconstruction (57% tibia,26% femur,2% fibula,4% ankle,7% humerus,4% knee). Results: Donor’s age of patients was significantly correlated to CD45/CD34 concentrations (R=-0.305,p=0.012;R=-0.353,p=0.003,respectively) on BMN (CD45 was also correlated to CD34 level,R=0.592;p<0.005). The level of CD34/CFU (in the BMC) was significantly correlated to the time required for BMN collection (R=-0.263,p=0.03;R=0.374,p=0.003, respectively). The concentration of CD34/CD45 in the BMN was significantly correlated to the BMN volume (R=0.392,p=0.001 and R=0.372;p=0.002, respectively). The Sepax© processing significantly influenced the CD34 (R=0.448,p<0.005) and CD45 (R=0.571,p<0.005) concentration found in the BMC. A significant reduction of CD34 (-43%)/CD45 (-44%) levels, the cellular viability (87% vs. 93%) was found in the BMC (in comparison to BMN,p<0.005). No microbiological contamination was found in both BMN/BMC. A mean of 65.2% of full consolidation was obtained while 15% did not succeeded after a mean of 17 months. Discussion: The composite graft made of BMC (with concentrated stem cells) supplemented with DBM (for osteoinduction) demonstrated that it is: (i) a clinical procedure compatible with cleanroom processing, (ii) a safe and efficient processing of BM, (iii) a significant improvement of bone non-union consolidation up to 84,8% (to recover a total or partial consolidation).

148

O-066 – Vascular homografts: life savers in transplantation and oncology surgery J. Lerut, N. Hetsch Starzl Abdominal Transplantation Unit CUSL-UCL Brussels

The modern development of surgery includes technical progresses and innovations also a widening of indications. This is especially well documented in the field of transplantation and oncologic surgery. Extension of indications frequently need the sacrifice of venous or arterial structures in order to make the planned surgical procedure possible. The replacement of such vascular structures should therefore be an integral part of the pre operative scheduling. Vessels can be replaced either by prosthetic material such as Goretex or Dacron or by vascular homografts. The procurement of free arterial and venous grafts should therefore become” an integral part of every donor operation. Already by 1979 TE Starzl demonstrated the successful use in the field of transplantation of inferior vena cava, portal vein, iliac veins and arteries in the field of kidney and kidney pancreas and liver transplantation. Carotid arteries, jugular veins, pulmonary veins and arterial mesenteric axe have been shown more recently to be very useful aids to solve some times difficult problems during different transplantation procedures. As the use of such free vascular grafts had been shown to be life saving, Starzl quoted that (liver) transplantation should never be attempted without an emergency assortment of such grafts. These procured free vascular grafts can be preserved in a UW solution to which antibiotics have been added. These vessels can be used up to 3 weeks after procurement. The need for such free arterial grafts has become even more and more important since the introduction of living donor transplant procedures. Especially in the field of liver transplantation it is very important in order to obtain an adequate liver function to restore as well inflow as outflow vessels. This can in many cases only be done by using vascular homografts. In the Asiatic countries such reconstructions are done with cryopreserved arteries and veins. More recently, the development of intestinal allo- and auto transplantation and also the development of wide “en bloc” resections for abdominal tumors have underlined the need to recur to the use of these vascular homografts. Every effort should be done from now on to obtain adequate vascular homografts in performing an organ procurement. Logistics should be set up adequately in order to optimize the preservation of such grafts. The set up of a cryopreserved vascular homograft bank becomes a necessity in order to further allow development of very difficult transplantation and oncologic procedures. Recovering such grafts should therefore be an integral part of every donor procurement. Logistics should be set up to handle different types of vascular homografts

149

O-067 – Development of Global Cardiovascular Nomenclature I. Uhrynowska-Tyszkiewicz,1 R. Jashari2 1 Medical University of Warsaw / National Centre for Tissue and Cell Banking, Warsaw, Poland

2 European Homograft Bank (EHB), Brussels, Belgium on behalf Joint ETTAG-NATTAG Terminology Work Group for Cardiovascular Tissues

Aim: The development of global cardiovascular nomenclature is one of the current aims of ICCBBA. Background: ICCBBA is an international, non-governmental, non-profit, information standard organization which manages the ISBT 128 standard - a global standard for the identification, labeling, and information transfer of human blood, cell, tissue, and organ grafts across international borders and disparate health care systems. The organization includes various technical advisory groups (TAGs) made up of 270+ volunteer experts. There are 3 TAGs which deal with tissues: North America Tissue TAG (NATTAG), European Tissue TAG (ETTAG) and Eye Bank TAG (EBTAG). Methods: The Joint ETTAG-NATTAG Terminology Working Group for Cardiovascular Tissues was established in 2011. To ensure as global approach as possible during the revision of existing ISBT 128 cardiovascular tissue graft terminology the experts not only from Europe and North America but from other parts of the world such as South America, Asia-Pacific region and Australia were invited to participate in the Working Group. The group works by consensus. Meetings are held through conference call and e-mail discussions. Results: To date, 14 individuals form 8 organizations were interested to offer their time and knowledge by taking part in cardiovascular project. They met 9 times by teleconference calls. A prepared consensus for class names of cardiovascular grafts and their definitions was a subject of public consultations between June and August 2013. Summary: After final adjustments the Cardiovascular Tissue Nomenclature will be public and published on the ICCBBA website as a part of in the standard terminology document. It is worthy to note that the established terminology can be used for any coding system, not only for systems using ISBT 128 standard.

150

O-068 – Arterial allograft bypasses in critical limb ischemia: a thirteen-year experience H. Chenorhokian, C. Perot, A. Bianchini, S. Amiot, D. Massouille, J. P. Chambon Autologous vein material is the preferred conduit for infra-inguinal bypasses. In absence of autologous vein material for below-knee bypass grafts, arterial allografts (AA) have been considered a viable substitute. Our study presents the results of a thirteen years experience using cryopreserved allograft in the treatment of critical leg ischemia Method: Between 2000 and 2013, 45 limbs with CLI on 41 patients were treated with below the knee bypass using an AA. There were 22 men and 19 women, median age 67 (extremes 35-87). The AA consisted in cryopreserved superficial femoral arteries and were supplied by the European Homograft Bank (Brussel, Belgium) Proximal anastomosis were performed on common femoral artery in 39 cases, prosthetic bypass in 3 cases, above the knee popliteal artery in 2 cases and deep femoral artery in 1 case. Distal anastomosis were performed on below the knee popliteal artery in 13 cases, tibio-peroneal trunk in 3 cases, posterior tibial artery in 6 cases, anterior tibial artery in 17 cases and peroneal artery in 6 cases. Patients were followed up through clinical examination and Duplex at 1, 3 and 6 month then yearly. Primary and secondary patency rates, survival rates and limb salvage rates were calculated by the Kaplan-Meier method. Results: One patient was lost to follow-up at 27 months. Median follow-up was 46 months (extremes 1- 140). 19 patients required major limb amputations and 9 patients died during follow-up. Primary and secondary patency rates were respectively 35% and 60% at 12 month, and 33% and 54% at 24 month. Limb salvage rate was 80% at 12 month and 67% at 24 months. 25 bypasses required a total of 45 secondary procedures to try to maintain patency. Amongst these procedures, 17 were performed for allograft degradation (14 stenosis, 3 aneurysm). Discussion: Below the knee bypasses using AA for patients with CLI appears to be a viable solution in absence of autologous vein. However, high rates of bypass thrombosis and allograft degradation require a strict follow up of patients and frequent secondary interventions.

151

O-069 – Treatment of aortic valve endocarditis with homograft: indications and long term outcome

G. El Khoury

OBJECTIVE Active aortic valve endocarditis (AVE) remains a challenge in terms of surgical strategy. Since 1989 we use a tailored approach to treat AVE. We report our surgical experience at 20years. METHODS Between 1989 and 2010, 229 consecutive patients underwent surgical treatment for active aortic valve endocarditis (AVE). Choice of valve substitute was determined by localization and extension of the infection, patient’s age and homograft availability. 73% had native AVE and 27% prosthetic AVE. In 135 patients (59%) aortic valve/root was repaired or replaced by a homograft/pulmonary autograft (human tissue: group I). 94 patients (41%) had a mechanical or biological prosthesis (prosthetic: group II). All homograft valves were supplied by the European Homograft Bank (EHB) in Brussels. RESULTS Mean age was 58±14 years. Patients in group II were older (63 vs. 55 years, p<0,001) and had more often heart failure (39% vs. 24%, p<0,05). Mean duration of follow-up was 88±76 months. Five year survival and freedom from endocarditis recurrence is respectively: 82±7% and 97±3% in group I vs. 60±10% and 90±7% in group II (p<0,05). Five year freedom from bleeding events was 96±4% in group I vs. 91±7% in group II (p=0,01). Main reason for reoperation was aortic regurgitation in group I and recurrent endocarditis in group II. After multivariate analysis human tissue was independently protective against endocarditis recurrence (HR=0,31; p=0,04), stroke (HR=0,15; p=0,01) and bleeding event (HR=0,36; p=0,02) CONCLUSION Surgical strategy in these complex situations depends on the extension of the infection, the age and the clinical state of the patient. When the infection is limited to the aortic leaflet, valve replacement with prosthetic valve, Ross procedure or valve repair procedure are good options. However, in the cases with extensive leaflet destruction, abscess formation and cases with septic state we prefer to use a homograft.

152

O-070 – Pulmonary homografts for right ventricular outflow tract reconstruction during Ross procedure: twenty years results A. Prat

Service de Chirurgie Cardiaque, Hôpital Cardiologique, CHRU de Lille, Lille, France

Background: Replacement of the aortic valve or aortic root with a pulmonary autograft (Ross procedure) is widely used for aortic valve disease in growing patients and young adults. In most cases, a cryopreserved pulmonary homograft is used for reconstruction of the right ventricular outflow tract (RVOT). Main drawbacks of this procedure are progressive dilatation of the pulmonary autograft and RVOT failure, the predominant indication for reoperation of the pulmonary conduit being stenosis. The aim of this study was to evaluate the long-term hemodynamic behaviour of pulmonary homografts in pulmonary position after Ross procedure. Methods: Four hundred and fifteen patients had a Ross procedure in our institution between March 1992 and June 2013. Among them, 348 patients received a cryopreserved pulmonary homograft (supplied by the European Homograft Bank) in pulmonary position and they represent the study population. Mean age was 28.2 ± 10.4 years. Mean homograft diameter was 25.8 ± 2.2 mm. A comprehensive echocardiography was performed at discharge, at 6 months and then on an annual basis. Pulmonary stenosis was defined as a mean transvalvular gradient of more than 20 mm Hg across the homograft. Median follow-up was 5.2 years (range, 7 days-20.5 years). Results: Perioperative mortality was 2.3 % (8 patients). Late mortality was 2.3 % (8 patients). During follow-up 10 (2.9 %) patients had reoperation on the RVOT with a mean time-interval of 9.0 ± 4.0 years. Three of them received percutaneous implantation of transcatheter pulmonary valve prosthesis 12.2 ± 1.7 years after the Ross procedure. Causes of reoperation were homograft failure in 5 cases and pulmonary endocarditis in 5 cases. In addition 5 patients developed a pulmonary valve endocarditis medically treated. Mean post-operative transpulmonary gradients were respectively 4.1±2.5, 8.6±6.3, 11.9±10.9 and 10.8±6.9 mmHg at discharge, 5, 10 and 15 years . Freedom from pulmonary stenosis was 99.0% (IC 95%; 98.3-99.7%); 97.5% (IC 95%; 96.2- 98.8%) and 88.6% (IC 95%; 88.2- 92.4%) at 5 and 10 and 15 years. Conclusion: Pulmonary homografts represent a safe valvular substitute for RVOT reconstruction during Ross procedure in our institution. This may be due to our policy of systematic oversizing of the allograft and the liberal use of anti-inflammatory drugs in the post-operative period. The gradual transvalvular gradient increase during follow-up however requires continued echocardiographic monitoring. Patients who meet criteria for isolated RVOT replacement can be successfully treated with catheter-based pulmonary valve implantation.

153

O-071 – Full thickness skin grafts: quantifying graft contraction following explantation L. Jares, C. Wells, J. Michael Real, L. Temple The Musculoskeletal Transplant Foundation

Background: Explantation of skin grafts utilizing the Full Thickness Skin (FTS) surgical approach has proven to be more efficacious in comparison to handheld dermatome use. FTS grafts are considered to contract or “shrink” following explantation more than dermatome recovered grafts due to the complete thickness of the papillary and reticular dermis. Anecdotal estimates regarding the amount of skin shrinkage that occurs following procurement are common and varied. Since skin banks seeking to obtain and process large, full thickness grafts are at the mercy of imprecise assessments during donor evaluation, a study that carefully quantifies rates of skin shrinkage will help allograft tissue recovery team members plan their surgical procedures appropriately. Purpose: The hypothesis when designing this study was that the square area of full-thickness skin specimens recovered from living or deceased donors (not using a dermatome) contract at least 10% between in situ (prerecovery) and processing measurement. Methods: Two busy recovery agencies in the United States (Donor Network of Arizona in Phoenix, Arizona and Midwest Transplant Network in Kansas City, Missouri) were selected based on donor volume and technician skill level and began participation following training on the study protocol and receipt of sterile supplies. Each donor was marked on the posterior torso by using two semi-rigid, sterile rectangle shaped templates of known sizes (measuring precisely 20 cm x 30 cm and 12 cm x 22 cm respectively) prior to surgical incision. Both templates were applied to the skin and carefully outlined by the recovery technician using a sterile, indelible marker. Upon receipt into the processing facility and prior to any tissue processing, both marked outlines on the epidermis were measured on all sides to the millimeter. Measurements were recorded and supplied for comparison to known pre-recovery markings Results: The donor sample size consisted of 70 deceased skin donations received during an 82 day time period. Donor ages range from 16 years to 60 years with a median age of 50 years. Of the 70 cases, 67 had both rectangle markings in measurable condition leaving three cases where only one rectangle was clearly measurable. Therefore, a sample size of 69 donors was marked clearly with larger rectangles and 68 were marked with smaller rectangles. The 69 “large rectangle” outlines, initially measuring 600 cm2, were measured at processing and averaged 516.8 cm2 (±102.3 cm2 at 2s; p value = 0.980). The average shrinkage rate equals -13.87% (-83.2 cm2). (Chart A) The 68 ‘small rectangle’ outlines, initially measuring 264 cm2, were measured at processing and averaged 223.9 cm2 with standard error (±42.3 cm2 at 2s; p value =0.896). The average shrinkage rate equals 15.19% (-40.1 cm2). (Chart A). This study would be lacking if we did not consider the distribution of shrinkage rates according to the variable of donor age. When subdividing shrinkage rates by donor age, the data supported the commonly accepted idea that skin shrinks less as donors grow older. This can be graphically illustrated by comparing donors at opposite ends of the data set. For instance, 3 donors, ages 15-19, demonstrate a mean shrinkage rate of 27.4% while another 20 donors, ages 55 – 60, averaged only an 8.1% loss in square area. (Table I, Chart B). Skin donations from living donors were also marked and measured for this project and the tentative findings based on the data set indicates shrinkage of -14.05% (-37.1 cm2 ± 9.2 cm2), however the sample size is too small (n=12) to draw any significant conclusions. Further data would need to be collected and evaluated to draw further conclusions and compare living and deceased data sets. Conclusions: The results of this study proved the hypothesis that skin from deceased donors

154

shrinks at least 10% with average shrinkage rates in this study of -13.87% and -15.19% (large and small rectangles respectively) and a maximum contraction of 32.7% (16y.o. female). On average, setting recovery parameters for full thickness skin grafts at 20% larger than the minimum acceptable standards of the receiving skin processor would negate tissue shrinkage on most donors over age 30. For example, a graft required to be at least 1200 cm2 at processing should measure at least 1440 cm2 prior to recovery incision. For donors less than age 30, this study indicates that grafts would have to be around 25-30% larger than minimum size standards due to significant, age related shrinkage. Although this study was small with n=69 donors and Normality Testing demonstrating predictability in the data set, a larger scale study would be helpful in confirming these conclusions. Finally, further study is recommended to establish whether skin contraction varies by body region or depending on the orientation of skin grafts. I.e., lateral versus inferior / superior skin shrinkage.

155

O-072 – ‘Activated Bone’ a novel treatment approach for large bone defects, even in infections; a Case Report K. Kaudela,1 W. Kaltenbrunner,2 J. Barth3 1 European Cell and Tissue Bank (ECTB), Wels, Austria 2 LK Zwettl – Gmünd - Waidhofen Orthopaedic Department 3 KH Branau, Department Traumatology, Orthopaedic and Sports Medicine

Introduction: Extensive bone defects caused by trauma or tumours usually require a very long treatment time with a high complication rate, massive reduction of life quality and a high social and economic burden. Goal: Platelet derived growth factors have been shown to stimulate cell proliferation effectively in vivo and in vitro, the trophic effect has already been tested in several trails for tissue engineering and regenerative therapy. Varying efficiency is considered to be at least in part due to divergent concentrations of growth factors. We were using a pool of human allogenic platelet lysate (p HPL), derived from routinely produced platelet rich plasma (PRP) of fourteen to fifty single blood donations. In addition to numerous factors it contains high concentrations of IGF, PDGF-BB, TGF-b and FGF-2. We impregnated a CO2 purified allogeneic human 18 cm long femur diaphysis (eCOO technology), which was demineralized about 2-3 mm depth, with this pHPL and with Vancomycin and freeze dried it. Material and Methods: A 23-year-old woman acquired a 3° open femoral fracture by a motorcycle accident. After initial treatment with an external fixator and recovering of the soft tissue conditions intramedullary nailing was performed. As there were no signs of callus formation after nine months several shock wave treatments took place and finally a revision with local application of BMP's. Again no union of the fracture and additional infection developed. A stage revision with debridement, removal of 16.5 cm diaphyseal bone, and implantation of 18 cm femur diaphysis, filling the intramedullary defects with 90 cc Tobramycin and Vancomycin impregnated allogenic spongy bone grafts (OSTEOmycin) and intramedullary nailing. Results: Patient could be dismissed on 10th post-op day with 25 kg weight bearing. Control after 4 weeks showed callus formation around the implant and at the docking sites, no signs of infection. After 8 weeks the patient is able to walk a few steps with full load, moving the knee joint freely again. Conclusion: ‘Activated bone’ seems to be a proper approach to address large bone defects, in combination with antibiotic impregnation technology (eCOO plus technology) even in infected cases. Author K. K. is the Responsible Person of ECTB.

156

O-073 – The use of human skin products in burn wound treatment T. Rose,1 G. Verbeken,1 S. Jennes,2 J.-P. Draye,1 J.-P. Pirnay1 1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology, Burn Wound Centre,

Queen Astrid Military Hospital, Brussels, Belgium 2 Burn Wound Centre, Queen Astrid Military Hospital, Brussels, Belgium The use of human donor skin and keratinocyte cultures has a very long tradition. They are used to cover large burns wounds and to stimulate and accelerate burn and donor site wound healing. Skin and keratinocyte banks are vital for successful burn wound treatment. The starting material of human (donor) skin products, as offered by public cell and tissue banks, is of variable ‘not standardized’ quality and inherently contaminated (at least with commensal bacteria). The end product is also whimsical and—as ‘product of nature’—difficult to protect by patents today. Biosynthetic dressings, in contrast, are business as usual for pharmaceutical companies. They can be produced from widely available raw materials, which can be used in GMP production, and the resulting end products are standardized, well defined and—last but not least—can be adequately protected by patents. To date, there are no biosynthetic skin replacements that provide the physical and physiological functions of human skin. Unfortunately, there are indications that industrially prepared biosynthetic dressings will replace human donor skin for the temporary covering of burns. The future of some of these lifesaving and established human skin products that are exclusively provided by public health institutions and small and medium-sized enterprises is threatened. We are convinced that these human skin products should remain the golden standard in burn wound treatment, at least until biosynthetic dressings reach an efficacy comparable to human skin products. We will show some examples of the clinical use of human donor skin and keratinocytes in our burn unit.

157

O-074 – Quality of life after the Ross procedure - reward for tissue bankers J. Spatenka,1,2 J. Vojacek,2 J. Burkert,1 M. Vobornik,2 P. Kobylka,3 A. Habrmanova,1 P. Zacek2 1 University Hospital Motol, Prague, Czech Republic

2 University Hospital Hradec Kralove, Hradec Kralove, Czech Republic 3 Institute of Hematology and Blood Transfusion, Prague, Czech Republic

Introduction: During last 70 years heart valve diseases surgical treatment moved from experimental labs to daily praxis. For 51 years the aortic and pulmonary allografts are routinely used, and for 46 years pulmonary allografts are utilized in Ross procedure as well. Clinical results of allograft heart valve surgery represent the principal stimulus for cardiovascular tissue bankers research with the aim to improve the grafts quality (mainly the long term performance). The patient’s life expectancy and the quality of life are the main targets of heart valves disease treatment. Goal: Aim of the study was to compare the quality of life of two institutional patients groups: First – 23 Ross patients, and second – 29 mechanical valve prostheses owners. Methods: Since 2009 the Ross procedure was performed in 28 patients. Perioperative mortality 0%, survival rate 100%, pulmonary autograft and pulmonary allograft function remains excellent. From that series of 23 patients (› 6 months after Ross operation) have been evaluated. The control group was formed from 29 patients (of comparable profile) who underwent the aortic valve replacement with mechanical valve prosthesis. The standardized RAND 36 questionnaire of Item Health Survey SF-36 was used for quality of life testing. Results: In the group 1, higher average values in all 8 quality of life categories were found. Significant difference was proved in categories of physical function (P=0.025), pain (P=0.030) and general health (P=0.002). Conclusion: On the basis of literature data and our own results (mortality, survival rate, pulmonary autograft & allograft function in follow-up, and last but not least the quality of life) the Ross procedure remains an alternative aortic valve replacement indication for active young and middle-aged patients in our department, mainly for those who must not/do not like anticoagulation. Despite the possible risks the quality of life in Ross patients was found to be unequivocally higher in comparison with a mechanical valve prostheses owners. These preliminary own results mean principal rewards for cardiovascular tissue bankers and research motivation to improve the grafts quality markedly.

158

O-075 – Factors influencing the survival of cryopreserved homografts B. Meyns

Cardiac Surgery, UZ Gasthuisberg, KULeuven, Leuven, Belgium

Objective: To determine the life span of cryopreserved homografts implanted in the right ventricular outflow tract and the factors influencing it. Methods: From 1989 through 2003, we reconstructed the pulmonary valve with 301 homografts in 272 patients (median age 13 years; range 4 days-69 years). Indications were tetralogy of Fallot (136), truncus (23), Rastelli repair (11), double outlet ventricle (13), endocarditis (5), and the Ross operation (84). Median follow-up was 5.7 years (range 0-14). We analyzed possible predictors of graft replacement by simple and multiple Cox regression. Results: Actuarial survival was 96+/-1.2% at 1, 95+/-1.4% at 5, and 94+/-1.5% at 10 years follow-up. Three homografts were explanted because of endocarditis (excluded from the analysis). Freedom from explantation was 99.6+/-0.4% at 1, 94.5+/-1.7% at 5, and 81.8+/-4.1% at 10 years. Variables, significantly related to explantation in the univariate analysis, were younger age, small graft size, implantation in a non-anatomical position, the aortic donor homograft, a shorter aortic cross-clamp time and the implantation of a second homograft. In the multiple model, non-anatomical position (P=0.001), smaller graft size (P<0.0001) or younger age (on square root scale, P<0.0001) and clamp time (P=0.01) remain as independent risk factors. Immunological variables, like blood group incompatibility, implantation of a second homograft and short warm ischemic time were not significant. Conclusions: The life span of a cryopreserved homograft is determined by graft size (correlates with age) and the non-anatomic position (correlates with indication). In a specific patient, the second homograft performs as well as the first.

159

O-076 – Current clinical experience with tissue-engineered allogenic matrices for pulmonary and aortic valve replacement S. Sarikouch

Cryopreserved homografts have been the gold standard for many years in selected indications such as for pulmonary valve replacement in congenital heart disease, severe bacterial endocarditis, or for right ventricular outflow tract reconstruction during the Ross -pulmonary autograft-operation. However, there is evolving evidence that tissue-engineered decellularized homografts may be superior to conventional cryopreserved homografts. At Hannover Medical School so far, 81 dezellularized, non-seeded, non-cryopreserved pulmonary homografts and 29 aortic homografts have been implanted with promising early to midterm results which are presented.

Figure1: Echocardiographic results 5 years after aortic valve replacement in a 8 year old girl.

Tissue-engineering of allograft matrices has left the laboratory bench and has become a valid certified alternative in pulmonary heart valve replacement, predominantly in the setting of congenital heart disease, and is gaining more and more clinical impact. The rising interest in these technologies is also reflected by an European Commission funded project on the application of regenerative heart valves which is aimed at translation of tissue-engineered allografts into widespread clinical use (http://www.espoir-clinicaltrial.eu).

160

Abstracts of poster presentations

161

P-001 – Bone allografts seeded with bone marrow derived mesenchymal stem cells and preosteoblasts – developing of a tissue bank procedure E. Olender,1 A. Kaminski,1 I. Uhrynowska-Tyszkiewicz,1 E. Urbanowska,2 A Wojtowicz3 1 Medical University of Warsaw, Department of Transplantology and Central Tissue Bank; National Centre for Tissue and Cell Banking, Warsaw, Poland 2 Medical University of Warsaw, Department of Haematology, Onkology and Internal Medicine, Warsaw,

Poland 3 Medical University of Warsaw, Department of Dental Surgery, Warsaw, Poland

Introduction: Recently a new approach to tissue grafts has been developed – using the allografts as scaffolds for cells and eventually as potential cell carriers for clinical application. Preparation of such type of combined grafts requires the involvement of a tissue bank/establishment. Goal: The aim of the study was to develop a safe and effective tissue bank procedure for preparing cell seeded bone allografts applied in mandibular defects augmentation. Methods: Trabecular bone allografts were prepared in form of 1x1x1 cm cubes under cleanroom class C conditions, irradiated with electron beam, 35 kGy and stored in -70°C. Bone marrow donors (20) were qualified according to tissue bank and hospital requirements. Cells obtained from bone marrow aspirates were either further cultured under GMP conditions and differentiated to preosteoblasts or directly used for revitalization of bone allografts. Cell viability was checked by a semi-automatic fluorescent method. Autologous preosteoblasts and autologous bone marrow derived cells were seeded onto bone allografts, placed in sterile wells and incubated in different conditions (media, duration and temperature). Seeded allografts underwent a quality control: PCRs for the expression of genes characteristic for osteoblastic differentiation and reference genes, and microbiological tests were performed, as well as histological sections. After incubation, seeded allografts were transported to hospital and implanted into mandibular defects to induce bone regeneration. Result: Microbiological tests performed on the incubation media revealed no bacterial/fungal contamination in each case. PCRs confirmed the presence of vital cells after incubation and the dependence of gene expression of the incubation conditions. Conclusion: A trabecular bone allograft revitalization tissue bank procedure was successfully worked out and clinically applied. Basing on this experience procedures for revitalization of other types of grafts are planned to be developed.

162

P-002 – Comparison of Cobe Spectra and Spectra Optia leukapheresis in relapsed glioma patients J. Dejaegher,1,2 D. Berckmans,1 F. Pauwels,1 L. Solie L,1 S. W. Van Gool,1,3 D. Dierickx,4 R. Smith,5 S. De Vleeschouwer1,2 1 Laboratory for experimental immunology, Catholic University Leuven, Belgium 2 Department of neurosurgery, Catholic University Leuven, Belgium 3 Department of paediatric oncology, Catholic University Leuven, Belgium 4 Department of hematology, Catholic University Leuven, Belgium 5 Terumo BCT

Introduction: Dendritic cell vaccination is a promising experimental therapy for glioma patients. Mononuclear cell harvesting by leukapheresis is the first step to generate dendritic cells. Spectra Optia (TerumoBCT) has the technical advance over Cobe Spectra (TerumoBCT) of a digital image capture system to automatically control the interface during centrifugation, and an additional collection chamber where the product is further purified from platelets. Goal: To compare our first Optia versus Spectra results. Materials and methods: 22 Optia and 96 Spectra collections were compared in non-cytokine-stimulated patients with relapsed high grade glioma after weaning of steroids. After leukapheresis, the Elutra system and identical laboratory techniques were used for generation of tumor protein loaded dendritic cells. Statistical analysis was done with unpaired T-tests. Results: The Optia and Spectra groups were statistically similar in terms of age, sex, length, weight and total blood volume. There were similar pre-leukapheresis measurements of RBC, WBC and platelets. After leukapheresis, Optia patients had lower RBC (11.1 versus 13.7%, p<0.05) and platelet (23.3 versus 39.3%, p<0.05) loss. The processed blood volume was less in the Optia group (10372 versus 11674ml, p<0.05), as was the collected volume (96 versus 133ml, p<0.05). The procedure took more time with the Optia device (222 minutes versus 197 minutes, p<0.05). In the collection, there was a lower concentration of contaminating RBC (0.26 versus 0.50x10¹²/l, p<0.05) and platelets (2227 versus 3022x10⁹/l) in the Optia group. The concentrations of WBC (98 versus 85x10⁹/l, p= 0.074) and monocytes (21.6 versus 17.1x10⁹/l, p= 0.078) were higher in the Optia group. Collection efficiency was higher in the Optia group (54 versus 45%), however this didn’t reach statistical significance (p=0.15). After elutriation and maturation, the final amount of viable immature (154.0x10⁶ Optia versus 152.0x10⁶ Spectra, p= 0.89) and viable mature (75.3x10⁶ Optia versus 68.1x106 Spectra, p=0.49) dendritic cells was similar. Conclusion: With the Optia system, less RBC and platelet contamination is seen, with similar monocyte recovery and dendritic cell yields from a smaller collection. The impact on dendritic cell function and clinical efficacy is yet to be defined. Author R. S. is an employee of Terumo BCT.

163

P-003 – Microbiological colonization of donor corneas after disinfection with PVP iodine J. Schroeter, F. Herrlinger, I. Wilkemeyer, A. Pruss

University Tissue Bank, Institute of Transfusion Medicine, Charité – Universitätsmedizin, Berlin, Germany

Introduction: Donor corneas are physiologically colonized with microorganisms and cannot be sterilized without destruction of their biological functions. Therefore disinfection and antibiotic treatment of the tissue are necessary. Methods: Conjunctiva and intraocular swabs were taken from 60 cornea donor eyes after periocular skin disinfection with 7,5% PVP iodine and ocular surface disinfection with 2% PVP iodine solution. After rinsing of the cornea and conjunctiva with 0,9% NaCl solution an in situ corneoscleral disc excision was performed and the donor corneas were put into organculture. The swabs were incubated in Thioglycolat solution for a maximum of 14 days at 32°C. Results: A microbiological colonization was found in 51 of the 60 donor eyes (85%). 106 out of 240 swabs showed a microbial growth. In contrast only 2 donor corneas (1.7%) showed microbiological contamination of their culture medium. In 27 of the 51 donor corneas which showed microbial colonization the microorganisms found were of higher potential virulence. None of the corneal grafts led to an infection of the recipient eye. Conclusion: The rate of microbial colonization of donor corneas is high, while the rate of contaminated culture medium is low. It remains questionable that a microbiological test of the ocular surface before donor cornea explanation can help reducing the risk of infections, especially endophalmitis in the recipient.

164

P-004 – Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts R. Díaz Rodríguez,1 B. Van Hoeck,1 S. De Gelas,2 F. Blancke,2 R. Ngakam,1 Y. Fan,1 R. Jashari1

1 European Homograft Bank, International Association, Brussels, Belgium 2 Laboratory for Control and Analysis (LCA), Brussels, Belgium

Introduction: The solvent dimethylsulfoxide (DMSO) is a common cryoprotectant solution for cryopreserved cardio-vascular allografts. Following the European Pharmacopoeia, DMSO is classified as a class 3 or low toxicity solvent. However, the determination of the residual level in the tissue is a mandatory part of the quality control of the allografts. Aim: To determine the residual DMSO in the cardiovascular allografts after thawing and their preparation for implantation. This study is carried out conform guidelines of the European Pharmacopoeia. Material and Methods: Four types of EHB allografts (aortic valve- AoV, pulmonary valve- PuV, descending aorta- DA, and femoral artery- FA) are cryopreserved using 10% of DMSO in Hanks medium 199. Sampling is carried out in three critical points of the procedure: after thawing, after total DMSO dilution and after delay of 30 minutes (estimated delay between thawing and allograft implantation). Thawing procedure is performed by progressive allograft defrosting in sterile warm water bath at 37-40°C. DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of physiologic solution. Finally, tissues are transferred to 200 mL of new physiologic solution during 30 min. Allograft samples are homogenised, centrifuged and analysed for residual DSMO concentration measurement using a standard extraction method and validated Gas Chromatography analysis. Results and Discussion: This research will provide valuable information regarding the residual DMSO content in allografts following the European Pharmacopea guidelines. The results of the analysis will allow the improvement of cryopreserved allograft thawing and diluting protocol in order to avoid the residual DMSO toxicity and, hence, improving quality life of patients.

165

P-005 – Inside a tissue bank QMS: Validation of transport and storage of Human Body Substances (HBS) for allograft preparation R. Ngakam, H. N. Akanyi, B. Van Hoeck, R. Jashari

European Homograft Bank (EHB), International Association, Brussels, Belgium

Introduction: Quality of cardio-vascular tissues is highly dependent of HBS’s storage and transport conditions while being transported from the procurement center. The transport conditions of HBS from procurement until processing in the tissue establishment must be stable and accurate (i.e. sterilely, at a temperature between 0 and 8°C, for 24 h max.). Aim: To ensure, taking into account the worst case scenarios (transport and storage below 0°C or above 30°C), that the materials and procedures used daily are able to ensure the latter 2 conditions. Procedures consist in putting the box or bag containing HBS and physiological solution inside 2 different types of insulated polystyrene boxes (coefficient of thermal conduction = 0.034 W/m.K) containing ice: a big one (volume 31.07 L; thickness 3.2 cm) and a smaller one (20.45 L; thickness 3.9 cm). Methods: The theoretical amounts of ice needed in each chosen scenarios (transport/storage round -18°C, 5°C, 24°C and 30°C) to keep the HBS within the fixed limits have been estimated taking into consideration polystyrene thermal conduction coefficient, heat flow through boxes walls and a given security coefficient. Experiments were then conducted to ensure the accuracy of these estimations by simulating at least 3 times the transport/storage in the different selected conditions and using temperature recorders to measure the HBM temperature. Results: Estimations and experiments showed that following amounts of ice were needed in the big box to keep the HBM within temperature limits during 24 h: 0.6 kg round 5°C (transport and storage), 3 kg round 24°C and >5 kg at 30°C. In the small box, those amounts were respectively reduced to: 0.5 kg, 1.5 kg and 2 kg. Experiments also showed that at -18°C, the 2 types of boxes were unable to keep the HBM within the fixed limit (temperature<0°C). Conclusion: While using minimum 2 kg of ice and a small box, it is possible to keep the HBS within given limits during 24 h in all storage/transport conditions except at -18°C. In case of a big box, amount of ice should be minimum 3 kg and only for storage/transport in a freezer (5°C) or at room temperature (24°c). This shows the importance of transport boxes selection.

166

P-006 – Validation of cardiovascular tissues packaging system in compliance with ISO 11607-2 recommendations

R. Ngakam, H. N. Akanyi, B. Van Hoeck, R. Jashari

European Homograft Bank (EHB), International Association, Brussels, Belgium

Introduction: Packaging system is very important for the cryopreservation and distribution of cardiovascular tissues. Its purpose is to protect and ensure product’s sterility during storage period until utilization. Due to the critical importance of this system, it has to be validated according to European directive 2006/86/EC in each unit producing sterile products and ensuring their sterility until utilization with this system. In EHB’s case, this mainly leads to assess the ability of welding devices (i.e. 2 devices for the external pouch and 3 for the internal pouch) to properly seal the 2 pouches that constitute EHB’s packaging system. These pouches were chosen in order to fulfil the requirements of ISO 11607-1: internal = EVA pouches, external = trilaminate pouches. Methods: Few pouches were sealed using aforementioned devices under ordinary working conditions, separated in 4 groups (just after sealing, 1 hour after sealing and storage in liquid nitrogen vapor, after 1 week, than stored at the same conditions, and after 1 week inside liquid nitrogen) and tested by:

- Visual inspection and peel testing to ensure pouches non-delamination and absence of holes in the sealing line;

- Leak testing using ink to ensure absence of grooves, tears or holes inside the sealing; - Resistance testing to assess sealing ability to resist pressure increase (the seal must

withstand a force greater than 1.5 N/15 mm welding according to ISO 11607-2).

Results: Results obtained show that all devices were able to seal the pouches without damaging them (no groove, tear or hole). Also, they were able to withstand a force greater than 19.62 N/15 mm welding for more than 15 minutes. On the contrary, one of the devices used to seal internal pouches showed very bad results at the ink test (leaks for the 4 different groups of pouches). Conclusion: One of the devices used was not reliable and should be repaired and retested before further utilization in order to ensure the sterility of cardiovascular tissues until implantation. On the other hand, effects on the sealing of the storage at very low temperature are minimal in this case.

167

P-007 – An international survey of tissue banking P. Myint,1 J. Wondergem,2 Y. Pynda,2 G. O. Phillips3

1 Oswestry Tissue Bank, The Long Barn, Chirk, Wrexham, United Kingdom

2 Applied Radiation Biology and Radiotherapy Section, Division of Human Health, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna, Austria 3 Glyn O. Phillips Hydrocolloid Research Centre, Glyndŵr University, Wrexham, United Kingdom

International Atomic Energy Agency has carried out a survey on the tissue banking activities from the tissue banks worldwide. The purpose of the survey is to identify the current tissue banking practices and to establish regional and global overview of the tissue banking activities so that the International Atomic Energy Agency can device further strategies to support tissue banking activities with particular focus on the use of radiation as a mean to sterilise tissues for transplantation. A questionnaire was sent out electronically to tissue banks that use irradiation or have a potential to use irradiation as a method of sterilising tissues. Eye banks were excluded. The questionnaire includes multi-sections about donation, processing, facilities and quality systems as well as R&D activities. Around 60 tissue banks of various sizes submitted the data with varying degree of activities in the number as well as the type of tissues processed. Majority of tissue banks are part of the hospitals or government institutions, hence determine the nature of the activities being performed. The approach to setting sterilisation dose varies from place to place resulting in different irradiation doses being applied. The tissue banking globally shows healthy and increasing level of activities supported by activities such as training, public awareness of tissue donation and training. The survey highlights the differences in processing methodologies among the organisations, the regional variations and some global trends in tissue banking. It poses a challenge to tissue banking community towards harmonisation of procedures and practices as the tissue banking activities continue to grow.

168

P-008 – Cellular grafting for bone healing V. Nacu, D. Vartic, V. Razborschii, P. Ciobanu Laboratory of Tissue Engineering and Cellular Cultures, State Medical and Pharmaceutical University ‘Nicolae Testemitanu’, Republic of Moldova

Introduction: Repair of bone defects, and also non-unions remains a major concern in bone reconstructive surgery, because of: limited supply of autograft, donor site morbidity, risks and complications in allografting and synthetic bone substitutes. It was identified that cord blood, like bone marrow, is a rich source of stem cells including hematopoietic stem cells, mesenchymal stem cells and embryonic-like stem cells. The aim of presented study was to evaluate the grafts containing allogenous cord blood cells or autogenous bone marrow cells with allogenic demineralized bone to stimulate bone formation in nonunion and pseudartroses. Methods: Materials used: allogenous demineralized bone matrix, autogenous bone marrow, Umbilical Cord blood stem cells, cultivated cells. Methods: biochemistry, cells differentiation, X-ray, Computed tomography. Results: This prospective study which was done on 175 patients with long bones non-unions. The Upper limb bones(61): clavicle – 3, the arm - 17; the radial bone - 9; the ulna - 7; the both bones of the forearm – 8, the scaphoide bone of the wrist – 14, the flanges - 3. The Lower limb bones (114): the femoral neck – 3; the femoral bone - 35; rotula -2, the tibial bone -74 cases. The results were evaluated by X-Ray, scintigraphy and by CT every month until the bone healing. In 16 cases as a grafts were used allogenous cord blood cells and the rest (159) autogenous Bone marrow stem cells. The bone healing were obtained in 82,1% of cases, in the rest of the cases patients needed surgical methods of treatment with autogenous cancelous bone grafts. Conclusions: The used cells grafts were compatible with the recipients, provides active and uniform osteogenesis in to the bone non healed site.

169

P-009 – Preliminary outcome of using cryopreserved human amniotic membrane in primary pterygium in Libyan patients N. Marsit,1 N. Gafud,2 E. Kafu,2 A. Tawil,2 A. Mabrouk,1 E. Abdalla,1 M. Fellah1 1 Tissue Banking Research Group / Biotechnology Research Center (BTRC), Tripoli, Libya 2 Tripoli Eye Hospital, Tripoli, Libya

Purpose: Amniotic membranes AM prepared at the Biotechnology Research Centre BTRC tissue bank were used in ocular surface reconstruction at the Tripoli Eye Hospital to assess their safety and effectiveness. Methods: Thirty-four cases operated for pterygium excision and amniotic membrane transplantation AMT, the surgeries were carried out during the period (September 2012 to March 2013), and the range of patient’s age is (20-75 years) with mean age of 50 years. AMT is indicated when other treatments is fail to prevent pterygium recurrence i.e. bare sclera excision. Results: The preliminary results of the surgeries showed that (94.1% cases) has no intra-operative complications nor post-operative complication after six months follow up. Conclusion: The primary pterygium excision with AMT using cryopreserved human AM is safe and effective surgery with low recurrent rate. None of the authors have any proprietary or financial interest to disclose.

170

P-010 – Use of allogenous bone graft and osteosynthetic stabilization in treatment of massive post-sternotomy defects M. Kalab,1 J. Karkoska,2 E. Matejkova,2 V. Lonsky1 1 Department of Cardiac Surgery, University Hospital and Faculty of Medicine, Palacky University, Olomouc, Czech Republic 2 National Cell and Tissue Center, Brno, Czech Republic

Introduction: Sternal dehiscence with massive post-sternotomy defects is a life threatening complication after the primary cardiothoracic operation. Mortality of this stage exceeds 70 %. Goal: Transverse plate fixation of the sternum is a currently used method of treatment of sternal dehiscences. Application of transverse titanium plates enables a safe restoration of the chest wall stability. However, a massive deficiency in bone tissue of sternum and ribs often places limits to its use. Main goal of this study is to explore a new approach for achieving the maximal stability of the chest wall. Methods: Capitalizing on orthopaedic surgery experience, in six cases we replaced a massive deficiency of chest skeleton by an allogenous bone graft (a calva bone graft in the first case, sternal bone grafts in the five following cases) and applied a transverse plate osteosynthesis to achieve stabilization of bone grafts and chest wall. We have used bone grafts prepared by the tissue bank according to Czech legislation and EATB guidelines. Appropriate serology tests of donors for HBsAg, HCV, HIV-1, -2 and Treponema pallidum are always made. The graft is stored in the freezer at -80°C. Before the implantation, the grafts underwent a final de-frosting procedure at 4–6°C for 12 hours. In the operation theatre, 0.9% NaCl solution with neomycine was applied. We always performed a prophylactic resection of the residual skeleton edges 1 or 2 cm to the healthy bone tissue. A transverse plate fixation of the chest wall was applied. Results: A clinical and CT check-up examination 6 months later showed excellent results proving the wound healed and stability of the chest wall achieved in five cases. A decease occurred 36 days after the graft transplantation due to a hypostatic bronchopneumonia in one case. However, the autopsy report demonstrated a clinically healed and fixed chest wall. Conclusions: Allogenous bone graft transplantation could be a novel approach for achieving the maximal stability of the chest wall in the management of complicated sternal dehiscence. In our experience, two principles are paramount: an early radical tissue debridement and a strict repeated microbial examination of osseous fragments of the wound.

171

P-011 – Human tissues and cells: application by private clinics and oral implantologists in The Netherlands M. S. E. Bergers, M. J. Happel, P. Y. Zijlker-Jansen, A. G. Bokhorst

TRIP Dutch National Hemovigilance and Biovigilance Office

Introduction: The National Hemovigilance and Biovigilance Office TRIP performed a survey of the use of human tissues and cells by private clinics (private hospitals) and licensed implantologists. This survey aimed to complement information from the hospitals in order to obtain a comprehensive overview of institutions that apply allogeneic human tissues and cells subject to the EU Directives 2004/23/EC and 2006/86/EC on quality and safety of human tissues and cells. Method: Based on the Dutch Healthcare Register, the Private Clinic Quality Register, search engine Google and a list provided by the Healthcare Inspectorate, TRIP compiled a comprehensive list of private clinics in The Netherlands. Clinics were included if they offered plastic surgery, orthopaedic surgery, oral and dental surgery, ophthalmology and ENT surgery. In addition the Register of Licensed Oral Implantologists was consulted. In all 620 addresses were contacted by letter with a questionnaire about use and source of human tissues and cells. Results: In total 401 responses were received (response 64.7%). In all, 64 clinics and implantology practices stated that they apply autologous, allogeneic or xenogeneic tissues and cells. Autologous tissues used in the same procedure and xenogeneic tissues (23 clinics and implantology practices) are outside the scope of the EU directives. The following applications of human tissues were reported: allogeneic bone tissue used by implantologists in the reconstruction of the jaw, allogeneic skin tissue for the covering of gingival recession and in the treatment of burns and allogeneic tendons for the reconstruction of the anterior cruciate knee ligament. Xenogeneic tissues are sometimes erroneously classified as human. This shows that the origin of applied tissues is not always clear. Table 1. Application of tissues and cells in The Netherlands (2013). Application of

tissues and cells

Private clinic Dental implantology

practice

Total

Percent

No human tissues

and cells applied

135 201 336 54.2%

Autologous tissues

and cells

4 11 15 2.4%

Allogeneic tissues

and cells

9 32 41 6.6%

Xenogeneic tissues

and cells - 8 8 1.3%

No answer ticked - 1 1 0.2%

No response 99 120 219 35.3%

Total 247 373 620 100.0%

Conclusion: The majority of private clinics and implantology practices do not apply human tissues and cells. However, 41 (6.6%) private clinics and implantology practices do apply allogeneic human tissues and cells. These clinics and practices will be included in the annual inventory of all Dutch hospitals and clinics for the provision of figures on use of human tissues and cells and number of recipients for the annual report to the European Commission.

172

P-012 – The influence of Transplant Procurement Management (TPM) training program in organ and tissue donation and transplantation M. Istrate, G. Paez, M. Manyalich TPM – DTI Foundation, Hospital Clinic de Barcelona, Barcelona, Spain

Introduction: Challenges in health care call for focusing on relevant training that increase knowledge, enhance innovation, boost scientific excellence and deliver better health care. Transplant Procurement Management (TPM) has been providing training on organ donation as well as tissue banking, advanced therapies and blood banking in compliance with the agreed professional requirements. Methodology: TPM offers blended learning programs consisting in online and face to face components that can be taken together or separately. Online training: The E-learning method involves the development of a flexible, interactive and bidirectional teaching and learning action. Synchronous and asynchronous learning tools are used. Face to face training: The face-to-face course is designed to reinforce knowledge through experience and facilitate the switch from theory to practice, and consists in theoretical lectures, seminars and workshops. A survey was developed assessing the impact of training on development of policies, practice, career choices, leadership, and knowledge dissemination and translated into five languages (Spanish, English, Italian, French, and Portuguese). Individuals who had participated in TPM training courses were sent a recruitment letter and were also asked to forward the link to individuals active in donation and & transplantation (D&T). Links were posted on Facebook and handed out at organ and tissue donation events. Respondents were asked to rate on a 1–5 scale the influence of trainings on 12 items to answer a Research question: What is the perceived influence of specialized training programs on career, collaboration, and skills in D&T? Institutional review boards at the University of Barcelona and Purdue University (USA) approved the study. Results: 1102 participants agreed to take the survey, 87% reported participating in a TPM course, out of which 95% selected TPM courses as most influential. Specifically, 98% reported influence on knowledge [score 4.45/5], 93% on technical [4.15] and communication [4.14] skills, 89% on attitude toward D&T [4.08], 92% on motivation to work [4.23], 91% on desire to innovate [3.98], 87% and 79% on ability to change D&T practices [3.85] and policies [3.51], respectively. Conclusions: Participation in TPM training courses has positive perceived benefits.

173

P-013 – New edition results of the Donation and Transplantation of Organs, Tissues and Cells Master Degree (D&T) C. Balleste,1,2 J. M. Segur,1 R. Casaroli,1 J. L. Pomar,1 M. Alsina,1 M. Istrate,2 G. Paez,2 M. Manyalich1 1 Medical School, University of Barcelona, Barcelona, Spain 2 TPM – DTI Foundation, Hospital Clinic de Barcelona, Barcelona, Spain

Introduction: The D&T Master degree is offered by University of Barcelona (UB) with the collaboration of Transplant Procurement Management (TPM). 2 paths are offered (Research & Professional) with specific modules and 3 common modules: ‘Organ and Tissue donation’, ‘Organ Transplantation’ and ‘Tissues, cells & hematopoietic

progenitor’s transplants.’ The Master has been designed in Blended learning system (online + face to face) to ensure flexibility and a minimum of 8 face to face weeks. Goal: Evaluate the efficiency of the teaching programme through student’s feedback analyses and results. Methods: We analysed the following aspects for each branch specialization and from the total: students profile; participation and grades obtained (from 0 to 10); personal benefits from participation and lecturer’s evaluation (from 1 to 5); Research Projects and Final Master Reports. Results: Participant’s profile: 19 participants (8 in Research & 11 in Professional), with a heterogeneous cultural and professional profile background: 13 Medical Doctors, 14 Nurses and 2 Biologists, from 12 nationalities. 2 left for personal reasons and 17 did graduate. Internal

subjects evaluations: The evaluation showed the following scores: Content of the materials (4,59±0,19); presentation (4,53±0,21); Questions/answers minutes (4,56±0,25); Personal benefits (4,25±0,32). Common modules results: The evaluation was a summary of student activities and the tasks fulfilled by them: Organ and Tissue Donation On-line (8,09); Tissue Banking On-line (7,95); Organ Transplantation (8,3). Final Master Dissertation: The Research Path participants presented 7 Research Projects (4 on Donation, 2 on Transplantation and 1 on Tissues and cells) and the Professional Path participants presented 10 Final Master Reports (7 on Donation, 1 on Transplantation and 2 on Tissues and cells). The mean scores obtained were 8,45 for Research and 7 for Professionals. Conclusions: The results obtained in 2012-13 achieved the expectations and overcame last editions. This blended and modular methodology applied in this UB Master degree, has demonstrated that is able to offer the possibility to improve and practice research or professional skills in this field depending on participant’s goal.

174

P-014 – Donor reconstruction C. Braun, F. Kriner, L. Roessel, M. Graw Institute of Legal Medicine Munich, Munich, Germany

While in scientific discussions aspects in donor recognition, such as contacting relatives and the questions of contraindications, are usually addressed, the topic of donor reconstruction is only rarely mentioned. However, working with musculoskeletal and skin donation, the reconstruction of donors is one of the most important tasks for various reasons: Naturally the viewing of the body is very important to the relatives. It is relevant to discuss the family's consent taking into consideration their wish for a burial with an open or closed casket. Since the deceased will be transferred to a funeral home after the tissue donation and reconstruction, there is usually no difficulty for the funeral ceremony arising from the donation. This is especially important for the contact between morticians and relatives: The experience of the mortician could well hinder or facilitate the discussion with the relatives concerning tissue donation. Furthermore, the best possible reconstruction of a tissue donor is in our opinion an ethical requirement and sign of respect in itself. Additionally, not only ethical aspects have to be considered, but the requirements in burial laws and regulations also have to be taken into account. The Institute for Legal Medicine Munich has a long history of tissue donation and explanation for scientific and therapeutic purposes. We present our reconstruction techniques and experiences.

175

P-015 – Bioethics as a discipline of the skin donation F. Martinez-F., A. Barrera-L., K. Guzman-M., S. Ustoa, P. Vizcaino, C. Lomeli-R., B. Escobedo-G., J. A. Madinaveitia-V. Skin & Tissue Bank at the National Institute of Rehabilitation, Mexico City, Mexico

Background: The Bioethics was borne in 1971 by Van R. Potter as a discipline that combine biological knowledge and human values; including moral vision, decisions, behaviors and policies. The altruist donation of skin is a bioethical behavior, based on four bioethical principles: autonomy, beneficence, non-maleficence and justice. The altruist donation of organs and tissues is a therapeutic alternative that offers greater life expectations. However, this issue has faced different obstacles owing to the cultural concept of death. The altruist donation of human skin involving aspects of bioethics as human values, mainly for use of skin as therapeutic alternative for burns patients and secondly for unknowing family about the procuring “per se”. The family condition for altruist donation depends about many factors the mains considered here: prognosis of the patient, doctor-family communication, social worker and altruistic organization to report of the donation. In Mexico the Skin & Tissue Bank at INR considering the bioethical-cultural aspects giving during all procuring process an ethic behavior, based on information and orientation for each altruistic donor of human skin, including the dignification process. Conclusion: In many countries procuring of skin from deceased donor can be difficult to accept for the general public, because it is little known process, causing doubts that potentiate the family refuse. The information from procurement hospitals should ensure implementation of detailed norms of respect for the donor’s body (procuring), so as not to hurt feelings and ensure that external appearance is reconstructed as faithfully as possible. Acknowledgments: This research project is granted by the National Council of Science and Technology of México; Grant FOSISS/CONACYT-1-161624.

176

P-016 – The EU human cell and tissue legislation should overcome ethical issues J.-P. Pirnay,1 A. Vanderkelen,1 D. De Vos,1 J.-P. Draye,1 T. Rose,1 G. Verbeken,1 N. Ectors,2 I. Huys,3,4 S. Jennes,5 C. Ceulemans6 1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology (LabMCT), Queen Astrid Military Hospital, Brussels, Belgium 2 Tissue Banks, University Hospitals Leuven, KU Leuven, Leuven, Belgium 3 Department of Pharmaceutical & Pharmacological Sciences, Centre for Pharmaceutical Care & Pharmacoeconomics, KU Leuven, Leuven, Belgium 4 Center for Intellectual Property Rights, KU Leuven, Leuven, Belgium

5 Burn Wound Centre, Queen Astrid Military Hospital, Brussels, Belgium

6 Department of Behavioural Sciences, Royal Military Academy, Brussels, Belgium

The EC considers human cell and tissue products (HCT/Ps) to be tradable goods. This viewpoint raises relevant ethical issues such as excessive commercialization (e.g. import/export for profit and without regard for self-sufficiency), commodification and allocation for non-therapeutic applications (e.g. vanity procedures – Fig. 1). The EU HCT/P legislation disregards the above-mentioned ethical issues. EU policymakers adopted the principle of subsidiarity as a way of evading ethical issues. We feel that this is not appropriate when it comes to the field of healthcare, because one may assume that health is a universal ethical good. The EU should adopt a clear ethical position overcoming commercialization issues. We identified two main ethical principles applicable to the HCT/P transplantation field: the basic principle of ‘‘respect for human dignity’’ and the principle that ‘‘human bodily material should not be considered as a commercial product or a commodity.’’ The key question is: does the processing of human bodily material lead to a product that is no longer subject to these ethical principles? One could consider HCT/Ps to be ‘‘dual products,’’ consisting of human bodily material and an added value in the form of a technological process. Both parts clearly have a different moral status, which leads to an ethical dilemma; the human bodily material is not a tradable good, while the added technological process (knowhow) clearly is. The problem is that one cannot be sold without the other. A possible way out of this dilemma would be to use the ‘‘doctrine of double effect:” if an action has foreseen harmful effects practically inseparable from the good effect, it is justifiable if the following are true: • the nature of the act is itself good, or at least morally neutral; • the agent intends the good effect and not the bad either as a means to the good or as an end itself; • the good effect outweighs the bad effect in circumstances sufficiently grave to justify causing the bad effect and the agent exercises due diligence to minimize the harm. Translated to the HCT/P field, this could imply that the commercialization of human bodily material (foreseen harmful effect) could be justified when tissue establishments act in good faith and produce HCT/Ps for use in meaningful (e.g. life-saving) therapies (good effect in grave circumstances). The good faith of cell and tissue establishments could be reflected in a HCT/P cost price that only relates to the added technological process and this in a reasonable manner.

177

a) b) c)

Fig. 1. a) Human donor skin (HDS) is surgically removed using a dermatome. b) Use of HDS for temporary wound closure in life saving burn surgery. c) Use of collagen, extracted from HDS, in a lip enhancement procedure.

178

P-017 – Business oriented EU human cell and tissue product legislation will adversely impact Member States’ health care systems J.-P. Pirnay,1 A. Vanderkelen,1 D. De Vos,1 J.-P. Draye,1 T. Rose,1 C. Ceulemans,2 N. Ectors,3 I. Huys,4,5 S. Jennes,6 G. Verbeken1 1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology (LabMCT), Queen Astrid Military Hospital, Brussels, Belgium 2 Department of Behavioural Sciences, Royal Military Academy, Brussels, Belgium 3 Tissue Banks, University Hospitals Leuven, KU Leuven, Leuven, Belgium 4 Department of Pharmaceutical & Pharmacological Sciences, Centre for Pharmaceutical Care &

Pharmacoeconomics, KU Leuven, Leuven, Belgium 5 Center for Intellectual Property Rights, KU Leuven, Leuven, Belgium

6 Burn Wound Centre, Queen Astrid Military Hospital, Brussels, Belgium

The transplantation of conventional human cell and tissue grafts, such as heart valve replacements and skin for severely burnt patients, has saved many lives over the last decades. The late eighties saw the emergence of tissue engineering with the focus on the development of biological substitutes that restore or improve tissue function. In the nineties, at the height of the tissue engineering hype, industry incited policymakers to create a European regulatory environment, which would facilitate the emergence of a strong single market for tissue engineered products and their starting materials (human cells and tissues). In this paper we analyze the elaboration process of this new European Union (EU) human cell and tissue product regulatory regime—i.e. the EU Cell and Tissue Directives (EUCTDs) and the Advanced Therapy Medicinal Product (ATMP) Regulation and evaluate its impact on Member States’ health care systems. We demonstrate that the successful lobbying on key areas of regulatory and policy processes by industry, in congruence with Europe’s risk aversion and urge to promote growth and jobs, led to excessively business oriented legislation (Fig. 1). Expensive industry oriented requirements were introduced and contentious social and ethical issues were excluded. We found indications that this new EU safety and health legislation will adversely impact Member States’ health care systems; since 30 December 2012 (the end of the ATMP transitional period) there is a clear threat to the sustainability of some lifesaving and established ATMPs that were provided by public health institutions and small and medium-sized enterprises under the frame of the EUCTDs. In the light of the current economic crisis it is not clear how social security systems will cope with the inflation of costs associated with this new regulatory regime and how priorities will be set with regard to reimbursement decisions. We argue that the ATMP Regulation should urgently be revised to focus on delivering affordable therapies to all who are in need of them and this without necessarily going to the market. The most rapid and elegant way to achieve this would be for the European Commission to publish an interpretative document on ‘placing on the market of ATMPs,’ which keeps tailor-made and niche ATMPs outside of the scope of the medicinal product regulation.

179

Fig. 1. The human cell and tissue product transplantation field as devised by the EU.

180

P-018 – Collaboration with a national professional organization to develop continuing education for funeral professionals M. Haun,1 J. Mohr,2 K. Lotherington2 1 Canadian Blood Services, Ottawa, Canada 2 Canadian Blood Services, Halifax, Canada

Introduction: Funeral professionals indicate that 50% of all funerals are pre-planned, presenting a logical opportunity to initiate a donation discussion with clients. Goal: In 2008, governments gave Canadian Blood Services a mandate for organ and tissue donation and transplantation to support system performance improvement. In 2012 the Funeral Services Association of Canada (www.fsac.ca) was engaged as a national donation partner in the development of an on-line donation course targeted to funeral professionals to advance donation opportunities and improve relationships with recovery organizations. Methods: Experts in on-line learning solutions were retained to manage the development and implementation of the course. The course content was informed by a panel of five experts representing funeral home/medical examiner liaisons, an eye bank, a tissue bank, a provincial donation organization and an expert in organ donation. The course was developed over six months and launched June 2013. It has been implemented in both English and French and upon completion of the course participants can receive Continuing Education Units towards their ongoing funeral professional education. Results: Module 1 introduces the concept of dual advocacy and provides funeral professionals with knowledge and motivation to appropriately and positively raise the opportunity for donation during pre-planned funeral conversations and to direct clients to provincial donation organizations and donation intent registries to action their wishes or obtain additional information. Module 2 addresses the surgical aspect of organ and tissue recovery, encourages the development of collaborative working relationships with recovery organizations and provides guidance on expected practice of recovery organizations in body reconstruction. Links to all Canadian donation and recovery organizations’ websites and intent-to-donate registries are provided to facilitate and encourage communication and relationship development. Each course participant is asked to complete a course evaluation to compare their level of confidence in discussing organ and tissue donation with clients before and after completing the course. Conclusion: Professional education through on-line learning is one of many methods to engage funeral professional as partners in donation.

181

P-019 – Is the age a parameter to consider in skin recovery process? A. M. Savío, M. L. Pérez, A. Vilarrodona, O. Fariñas, E. Agustí, X. Alemany, S. Vito, R. Oliva, S. Luque, E. Trias Transplant Services Foundation (T S F), Barcelona, Spain

Introduction: The increment in the population of survivors among patients with large complex burns has forced to widen the sources where to obtain products able of covering the burned body surface area. Lack of autologous split-thickness skin graft for large complex burns requires the use of temporary cutaneous substitutes, being the skin from human deceased donors a good choice for that. Goal: This work aims to establish the relationship between the amount of square centimeters of suitable recovered skin and the age of the donor. Methods: This prospective and retrospective study was conducted on 419 human cadaver skin donors, available to the tissue bank during the period from January 2009 to July 2013. The relationship between the amount of suitable recovered skin and the age was determined. Different factors such weight, height, body surface area, body mass index, body cooling time before retrieval, type of donor, sex, associated diseases and recovery team leader were taken into account for the selection of an homogenous study group. Result: The donor average age was 58 years (range, from 13 to 84 years). Sixty five percent (65%) were male and (35%) female. According to the type of donor, 55 % were exitus donors, 25 % brain death donors and 20 % non-heart- beating donors. Mean body cooling time before retrieval was 4 hours. More than a million cm2 of suitable skin were recovered, of which, 38 % were cryopreserved and 62 % were glycerol-preserved. In accord with our study age was not correlated to the amount (cm2) of suitable recovered skin. Conclusion: The evidence obtained leads us to conclude that old donors can be as good skin donors as young ones.

182

P-020 – Impact of the national organ donation program on the tissue procurement promotion: a single study X. Merny, L. Lekeux, V. Vandenschrik, D. Vandeynse, D. Dufrane Cliniques Universitaires Saint-Luc, Brussels, Belgium

Introduction: Human donation remains the major limitations of the widespread use of clinical tissue applications. Goal: The work aims to assess the impact of the Belgium national "Gift" project on the tissue donation in our university hospital. Materials/Methods: The GIFT program was initiated in 2008 in our institution for organ donation. This first objective was a simple description of the potential organ donors from intensive care associated to the promotion of organ donation among medical/nursing staff. Secondly, a local coordination for identification/management and referencing of potential organ donation was initiated. In view to assess the beneficial effect of the GIFT program on the tissue donation, the course of tissue procurement was retrospectively analyzed among 264 human organ and/or tissue donors performed between 2006 and 2012 in the university clinical hospital Saint-Luc, Brussels. Results: Prior the initiation of the GIFT (2006/2007), means of 14 and 19 organ/ tissue individual procurements were annually performed. Among the organ donation, 13% of donors were referred to the tissue bank by transplant coordinators and 46.4% of organ procurements were followed by tissues procurements. Between 2008-2012, a significant increase of tissue procurements was reported at a mean of 24.6/year in contrast to any modification for the organ procurement (15/year). However, a significant decrease of tissue donors (referred by organ coordinators) by 6.1% was found during this period. In contrast, the number of tissue donors issues from organ procurements increased by a mean 14.8%. It was not also found any significant increase of tissue donation without referring from organ coordinators. Despite the development of tissue procurement in our institution, a large number of potential donors is not identified from intensive care units (72 and 57 potentials donors in 2011 and 2012, respectively). The causes of non-procurements remain: 86% (miss-identified) and 14% by refusals of family (10%), unavailable contacts families (3%) and parquet objections (1%). Conclusions: Although the GIFT program was initially implemented to improve the identification of organ donation/procurement alone, its major beneficial effect was observed for tissue procurement (15 vs 23 tissues donors alone in 2006 vs. 2012, respectively). Therefore, a similar ‘Tissue Gift’ program could be beneficial to potentiate the tissue procurement in terms of bone/tendon/skin/cornea...

183

P-021 – Evaluation of different microbiological testing methods in tissue banking J. D'Amato Tothova, L. Giurgola, C. Gatto R&D Department, Al.Chi.Mi.A. S.r.l., Ponte San Nicolò, Italy

Introduction: Each tissue bank validates its own method for microbiological analysis of tissues, which is essential to determine whether they can be released for transplantation. Purpose: To compare the results of microbiological analyses of tissues intended for transplantation obtained by using different microbiological testing methods. Material and methods: Human cardiovascular tissues, skin and corneas were retrieved and processed by five different tissue banks. Tissues were decontaminated at 4°C for 24h/72h (cardiovascular) or at 22°C for 90 min. (skin) either with bank prepared solutions or BASE.128 (AL.CHI.MI.A. S.r.l., Italy) and then cryopreserved in RPMI 1640 with the addition of 10% DMSO. Corneas were processed and stored under organ culture conditions. Microbiological analysis of tissues and processing liquids were performed by tissue banks according to their standard procedures, using BACT-ALERT, BACTEC and direct inoculation of culture media (Thioglycollate/TSB); samples were tested in parallel at AL.CHI.MI.A. with the sterility test according to European Pharmacopoea (EP), with the removal of antibiotic residues with the ResEP device. Results: All bacteriological analyses of decontaminated cardiovascular tissues performed with direct inoculum method resulted negative. 3% of liquid and tissue samples were found positive with the BacTEC method. The percentages of positive liquid and tissue samples with the ResEP device were 16% and 25%, respectively. The bacteriological analysis of decontaminated skin samples were negative with both BacTEC and Direct Inoculum methods. 33% of liquid samples and 50% of tissue samples were found positive using the ResEP device system. Corneas processing liquids resulted negative with both BacTEC and Direct Inoculum method. Instead, 59% of the samples resulted positive with the ResEP device and sterility test according to EP. Conclusions: Different results were obtained depending on the used method to perform microbiological analyses after tissue decontamination. A validation of the microbiological method, including accurate removal of possible antibiotic residues in decontaminated tissue and processing liquid is necessary to ensure the safety of tissue allografts. Authors J. D., L. G. and C. G. are employed in the R&D department of Al.Chi.Mi.A. S.r.l., the Company that manufactures the products discussed in the present abstract.

184

P-022 – Validation of ‘ResEP tube’ system for microbiological analysis with removal of antibiotic residues L. Giurgola, J. D'Amato Tothova, C. Gatto R&D Department, Al.Chi.Mi.A. S.r.l., Ponte San Nicolò, Italy

Introduction: Residual antibiotic concentrations may induce bacteriostasis of microorganisms eventually present on tissue samples and lead to false negative results during microbiological analysis of the sample. Purpose: To validate a ResEP tube system for microbiological analysis that reveals microbial contaminations of solid and liquid samples with removal of antibiotic residues. Material and methods: The amount of antibiotics in 3-5 ml samples of four bank prepared antibiotic cocktails, including the BASE.128 medical device (AL.CHI.MI.A. S.r.l.), and cardiovascular, skin, amniotic membrane and corneal tissue samples was determined by HPLC and agar diffusion analyses before and after treatment with ResEP tube. Interference of the device with bacterial growth was evaluated performing the microbiological recovery test with inoculants containing 1-10, 10-100 and 100-1000 CFU of European Pharmacopoeia (EP) reference strains (S. aureus, P. aeruginosa, C. albicans, B. subtilis, A. niger and C. sporogenes).

Additional tests of the device were performed using different fluids and tissue samples provided by five different tissue banks. Results: Agar diffusion test and HPLC showed important antibiotic residues in liquid and tissue samples not treated with the ResEP tube; the amount of antibiotic residues varied depending on the antibiotic cocktail. Complete antibiotic removal from liquid and tissue homogenates decontaminated with BASE.128 was observed after ResEP tube treatment. Removal of antibiotics from bank-prepared antibiotic cocktails varied from 94-99%. For EP reference strains, growth was observed for all the tested inoculants. ResEP tube analysis of samples processed and provided by tissue banks showed significantly higher bacterial recovery as compared to the tissue banks microbiological methods. Conclusions: The ‘ResEP tube’ microbiological test system was validated for the detection of microbial growth in both liquid and tissue samples with the removal of antibiotic residues. The device resulted to be harmless for EP reference strains and showed higher sensibility as compared to currently used microbiological methods in tissue banks. Authors L. G., J. D. and C. G. are employed in the R&D department of Al.Chi.Mi.A. S.r.l., the Company that manufactures the products discussed in the present abstract.

185

P-023 – Improved cryopreservation of endothelial monolayers and rat vessels in modified TiProtec G. Pless-Petig,1 S. Knoop,1 K. Bortlik,2 B. Zatschler,2 A. Deussen,2 U. Rauen1 1 Universitätsklinikum Essen, Essen, Germany 2 Medizinische Fakultät Carl Gustav Carus, Dresden, Germany

The vascular cold storage solution TiProtec, designed for hypothermic (4°C) storage, has previously been identified as a suitable cryopreservation base solution for aortic endothelial monolayers and aortic segments using viability assays and histology, respectively. We now assessed a variant of the solution with a different ion composition and added functional assays. Monolayers of porcine aortic endothelial cells were cryopreserved in cell culture medium, TiProtec, and a modified TiProtec with a different ion composition, all with 10% DMSO at -0.1, -1 or -5°C/min. After storage at - 80°C for 18-24 h, monolayers were rapidly thawed and re-cultured in supplemented M 199 cell culture medium. Cell viability (propidium iodide uptake), cell morphology and density, metabolic activity (resazurin conversion) and mitochondrial membrane potential and morphology (MitoTracker green, tetramethylrhodamine) were assessed. Rat vessel segments were cryopreserved in cell culture medium or modified TiProtec at -1°C/min and functionality after thawing was assessed in a Mulvany apparatus. Compared to cryopreservation in TiProtec (37 ± 11% living cells) and cell culture medium (12 ± 5%), viability after cryopreservation in modified TiProtec was markedly increased (53 ± 12%; values assessed after 3 h re- culture). In the latter solution, very few detached cells could be observed and the monolayer was largely intact. Mitochondrial fragmentation was seen after cryopreservation in all solutions, but was almost completely reversible in modified TiProtec within 3 h of re-culture. After 24 h of re-culture, viable cell density had increased in cultures preserved in the modified solution, indicating cell proliferation. The superior protection of both, TiProtec and modified TiProtec was apparent for all cooling rates, best results being obtained with a rate of - 1°C/min. Functional experiments, i.e. vasoreactivity assessments, of cryopreserved rat vessels, confirmed the superiority of modified TiProtec over cell culture medium. In conclusion, modified TiProtec greatly improved cryopreservation of endothelial monolayers and rat vessels, offering a promising approach for the improvement of vessel banking. Author G. P.-P. received a traveling grant from Dr. F. Köhler Chemie GmbH, which holds a patent on TiProtec, also covering the modified solution used in this study.

186

P-024 – The use of allogeneic, biostatic skin grafts and amniotic membrane in burns treatment D. Hoff-Lenczewska, M. Kawecki, A. Klama-Baryła, W. Łabuś, J. Glik, M. Kraut, M. Nowak

Burn Treatment Centre, Siemianowice Śląskie, Poland

Introduction: Allografts were used for decades and are golden standard for comparison with other temporary substitutes. Allogeneic skin dressings well protect the body against bacterial infections, reduce the loss of proteins, fluids, electrolytes, reduce pain and accelerate the healing of wounds. Size of biostatic dressings enables covering of large surface wounds. In addition, biostatic dressings are easy to prepare and sterilize, and are characterized by the simplicity of the application. Goal: Discussion of the preparation and differences in the use of biostatic, allogeneic dressing of amnion and skin, resulting from the experience of specialist unit treating burns and chronic wounds. Methods: Gaining of allogeneic human skin takes place during multiorgan donation. Amniotic membrane was obtained from placenta during cesarean surgery. Skin and amnion were prepared in the Laboratory of in vitro cell and tissue culture and Tissue Bank in accordance with their own procedures. Result: Patients with extensive burns have transplantation of allogeneic skin after decompressive incisions, early tangential cuts and deep excisions of necrosis in order to stabilize the general condition and reduce the size of local complications. Easiness of application and low cost of operation, and a very good health and aesthetic effects (inhibition of fibrosis - less chance of scar formation) cause the human amniotic membrane is an alternative to other biological dressings in the treatment of burn wounds. However amniotic membrane cannot be used for deep burns. Conclusion:

1. Amnion is a good biological dressing characterized by many features of an ideal dressing. 2. The use of amniotic membrane makes sense in the case of first and second degree burns. 3. Allogeneic skin grafts are ideal wound dressing when the blood supply of the wound is not sufficient to take the free-thickness skin graft.

187

P-025 – Effect of gamma rays (G) and accelerated electron beam (EB) on compact bone collagen crosslinks: influence of defatting, radiation dose and irradiation temperature

A. Jastrzebska,1 E. Grazka,1 J. Marowska,1 G. Gut,1,2 I. Uhrynowska-Tyszkiewicz,1,2 A. Kaminski1,2 1 National Centre for Tissue and Cell Banking, Warsaw, Poland

2 Department of Transplantology and Central Tissue Bank, Medical University of Warsaw, Warsaw, Poland

Introduction and goal: Fluorescent enzymatic mature crosslinks of bone collagen – pyridinoline (PYD) and deoxypyridinoline (DPD) – are considered to stabilize the collagen network, whereas pentosidine (PEN) crosslinks, resulting from nonenzymatic glycation of collagen, are supposed to increase its stiffness and may contribute to the susceptibility of bone to fracture. As compact bone tissue grafts should provide a long-term support for remodeling, the purpose of our study was to examine the possible effect of different processing and terminal sterilization methods of compact bone samples on the profile of these three collagen crosslinks, using ultraperformance liquid chromatography (UPLC). Methods: Compact bone samples isolated from femoral shafts of 6 male donors (aged 46-67 yrs), defatted or non-defatted prior to irradiation, were irradiated with G or EB with two doses (25 or 35 kGy) at different temperature (ambient temperature or dry ice). Non-defatted and non-irradiated bone samples served as control. Next bone samples were pulverized under LN2, defatted, lyophilized and hydrolized in 6N HCl. Crosslinks were extracted by solid phase extraction (SPE) on Chromabond Crosslinks columns, then separated and quantified in a single run by UPLC with fluorescence detector. Results were expressed as crosslinks density in bone collagen after examination of collagen content by colorimetric assay of hydroxyproline in bone hydrolizates. Results: Irradiation of compact bone rings with G and EB resulted in decrease of collagen PYD and DPD content in most experimental groups, whereas, in contrast, increased PEN levels were found. PYD appeared to be more sensitive to irradiation-induced decay than DPD. No consistent effects of radiation source (G vs. EB) and irradiation dose (25 vs. 35 kGy) were observed. Defatting of compact bone samples prior to irradiation seemed to enhance radiosensitivity of PYD with no marked effect on DPD, while to promote PEN formation. No irradiation temperature effect on collagen crosslinks content was found, irrespectively of radiation source. Conclusion: Radiation sterilization-induced changes in compact bone collagen crosslinking profile are supposed to contribute to altering irradiated bone grafts mechanical properties.

188

P-026 – Osteoinductive activity of peracetic acid-ethanol sterilized xenogeneic bone for transplants M. Anastasescu,1 J. Zunino,2 C. Gutiérrez,3 V. Seija,3 G. Semiglia4 1 Bone Bank. Hôpital Erasme, ULB, Brussels, Belgium 2 Tissue Bank, INDT, University Hospital, School of Medicine, Uruguay

3 Microbiology Department, University Hospital, School of Medicine, Uruguay

4 Department of Surgery, University Hospital, School of Veterinary Medicine, Uruguay

Secondary sterilization of bone grafts represents a pivotal step in musculo-skeletal tissue banking procedures. Several different sterilizing methods for relevant pathogen microorganisms’ inactivation have been used in the past and most of them have been discontinued, either due to their toxic, carcinogenic and/or mutagenic effects. Others, were blamed to alter some biomechanic, biologic or remodelling properties of bone grafts. Gamma radiation is a very common sterilizing method for processed bone grafts, but is known to alter mechanical properties and at some degree, inductive and osteogenic properties of grafts. Peracetic acid-ethanol sterilization (PES) has proven effective inactivating a wide range of bacteria, fungi and viruses. In addition, PES does not alter bone morphology, nor structure. Moreover, some authors have demonstrated that PES bone allografts are osteoconductive. Nevertheless, scarce literature documents thorough research about PES processed bone’s osteoinductive and remodelling properties. Since 2003, the authors have been using a xenogeneic (bovine), antigen-extracted, processed, freeze-dried bone for transplants. Experimental and clinical results have shown that our bone graft retains BMP activity, is osteoconductive and remodels after grafting. Thus, to evaluate whether PES affects biological properties of implanted bone xenografts, we designed an in vivo experiment in mice for assessing their osteoinductivity, osteogenicity and remodelling capacity. Care of animals was performed according to the guidelines contained in the National Research Council (Washington, DC, 1996), and the experiment was approved by the Honorary Commission for Animal Research (CHEA) of our School of Veterinary Medicine. After having tested peracetic acid-ethanol (PAE) antimicrobial activity using pathogen bacterial strains-embedded bones, disinfected xenografts were heterotopically implanted in mice. New bone formation (ossicles containing bone marrow) around and within implants was assessed both by X-ray studies and histologic techniques, as well. Results thus showed that PAE-treated bone xenoimplants were osteoinductive and osteogeneic. PES could thus represent an alternative to other sterilizing methods that alter both biomechanical and biologic properties of processed bone grafts.

189

P-027 – Human skin cell banking from multiorganic donors R. Garcia-C.,1 H. Sandoval-Z.,2 C. Machuca-R.,3 K. Guzman-M.,4 A. Barrera-L.,4 F. Martinez-F.,4,5 J. A. Madinaveitia-V.2 1 Ministry of Health of Mexico, Mexico City, Mexico

2 National Institute of Rehabilitation, Mexico City, Mexico

3 Molecular Therapeutic Lab, UMIEZ-FES Zaragoza, UNAM, Mexico City, Mexico

4 Skin & Tissue Bank at the National Institute of Rehabilitation, Mexico City, Mexico

5 Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City, Mexico

Background: The coverage of burns with skinfoil allografts is the best treatment for burned patients. News approaches and strategies are focused to build biological and cellular coverage with heterologous cells to increase therapeutical options. The Skin and Tissue Bank of National Institute of Rehabilitation (INR), uses a model of recovery and storage of human primary cells derived from skin of multiorganic donors. Hereby, we present preliminary results of quality control and patterns of grow of cultured for cell for banking at the Skin and Tissue Bank of INR. Methods: Recovery of tissues after several hypoxia time (n=15) was performed following the current protocol of the skin and tissue bank. Tissue slices were subjected to enzymatic method for cells isolation. Cell culture was maintained at standard condition into the class-100 core facilities. Microbiological, molecular and cellular phenotype control was performed at passage 1 and before cryopreservation. Viability assay was based on the DCF method and was employed to evaluate the grow curve until passage 5. Quantitative analysis was performed using the LSMeta-100 software by Carl Zeiss (Germany) for Laser Scanning Confocal Microscopy (MCL). Results: Viable cells were obtained of different times of procurement after last beat: cells were typified and cultured for fibroblasts and keratinocytes in specific media formulation. The histological analysis was performed for each type of culture. The range of number of cells obtained was inversely proportional to the postmortem time. Conclusions: Recovery of viable cells from multiorganic donors until 7 Hours of Last beat, is possible. Obtained cells keeps proliferation capacity and cell phenotype conserved at passage 5. Cell banking derived from multiorganic donors are proposed as alternative for tissue engineering and represent a therapeutic option for use in burn treatment. Acknowledgments: This project is granted by the National Council of Science and Technology of Mexico trough the project FOSISS/CONACYT-11-1-161624.

190

P-028 – Gene banking from Multiorganic Tissue Donors F. Martinez-F.,1 H. Sandoval-Z.,2 R. Garcia-C.,3 J. A. Madinaveitia-V.2 1 Skin & Tissue Bank at the National Institute of Rehabilitation, Mexico City, Mexico

2 National Institute of Rehabilitation, Mexico City, Mexico 3 Ministry of Health of Mexico, Mexico City, Mexico

Introduction: BPyT of the INR is the first tissue bank of the Ministry of Health and actually is a leadership for tissue banking activities in México. Based on scientific protocols, several sub-banking process are developed from a donor for scientific research goals. Objective: Improves and developing protocols for DNA, Cell and Tissue Banking in México for therapeutic and research uses. Material tissues and methods: A multi-institutional network was constituted to identify potential donors. Traceability of process, GLP and GMP are implemented as rutinary process for quality control and safety for human skin allograft and cardiovascular tissues. Carefully screening and protocolized donors are focused to donate: 1) Skin allografts, 2) Cells obtained from allografts (fibroblasts, and keratinocytes); 3) Remained samples of whole blood, are processing for DNA banking, and 3). Supernatant of whole blood are kept to construct a serotec for proteomic studies and typified levels of hormones and growth factors. Results: Since 2010 a bank of cells are resulting for this program. 1 cm2 of sample tissue of each donor of skin graft since 2010, are subjected to cell culture and expansion in vitro. Cells are cultured until 5x107 cells and preserved in Nitrogen. Genomic DNA is processed by phenol chloroform protocol and maintained in TE/ethanol at -80°C. Serum samples of 1.5 ml and kept at -80°C. Conclusion: Viable cells are potentially obtained from qualified donors, with complete genomic and proteomic profile. Samples are destined for scientific research purposes to determine levels of growth factors and hormones. Acknowledgments: This project is granted by the National Council of Science and Technology of Mexico, FOSISS/CONACYT-11-1-161624.

191

P-029 – Preliminary report of a start-up program of fertility preservation in men with cancer M. E. Rendal-Vázquez,1 M. García García,2 O. Fernández Mallo,1 M. Carballal Rodriguez,2 I. Miguez Torres,1 M. J. López Piñón,2 T. Bermúdez González,1 N. Ponte Velasco,1 J. Sánchez Ibáñez1 1 Tissue Bank, CHUAC, A Coruña, Spain 2 Laboratory of In Vitro Fertilization, CHUAC, A Coruña, Spain

We present our initial experience since the beginning of 2013. Each sample was analyzed in a semiautomatic analyzer (SCA Sperm Class Analyzer) to determine if the sample was suitable or not for processing, and with which method applying criteria used by IVF laboratories for handling samples to optimize each sample. In all the samples defrost tests were performed using the SCA, inverted microscope and pentoxifylline tests when required. A total number of 33 patients were sent to our unit for sperm cryopreservation. The average age was 31,4 ± 8,3 years. The most frequent diagnosis was Hodgkin's disease (39%) with testicular cancer (39%). In all cases a SCA was performed in the fresh samples and in 4 cases an inverted microscope analysis was required, in 3 of them a complete azoospermia was observed and the sperm were not processed. The sperm’s average volume was 4 ml, with a median of 16,6 millions spermatozoa/ml, mobiles 40%, progressives 27,3%. In 10 cases a posterior sperm capacitation was required and in those cases a pearls processing was necessary, showing an increase both in the number of mobiles and progressives. In 20 cases a classic cryopreservation method was used. In all the cases a defrost test was performed, demonstrating the viability of the samples for performing ICSI (intracitoplasmatic sperm injection). The sperm’s average number decrease in 29% when a classic cryopreservation method was used and a decrease of 54% in the pearl method. In this method the decrease of mobile and progressive spermatozoa was increased. Conclusions: Almost 10% of the patients showed azoospermia. One third of the samples needed sperm capacitation. A routine defrost test allows us to check the viability or not of the samples. In our opinion this kind of program should consider from the outset the application of criteria of posterior utilization to optimize the number and volume of samples looking for the highest possible success rate with these patients.

192

P-030 – Understanding the molecular structure of normal and osteoporotic human bone A. Lobo Gajiwala,1 A. K. Singh,2 R. S. Teotia,3 N. Sinha,4 A. Kumar,3 J. Bellare,2,5 U. Samant,1 C. D’Lima1 1 Tissue Bank, Tata Memorial Hospital, Mumbai, India 2 Centre for Research in Nanotechnology and Science, Indian Institute of Technology (IIT), Mumbai, India 3 Department of Bioscience and Bioengineering, IIT, Mumbai, India 4 Centre of Biomedical Magnetic Resonance, SGPGIMS Campus, Lucknow, India 5 Department of Chemical Engineering, IIT, Mumbai, India

Introduction: A study using high-resolution solid-state NMR (SSNMR) spectroscopy along with SEM, SEM-EDS-Mapping and TGA was carried out on normal and osteoporotic human bone with a view to evaluating processing protocols and providing data for designing bone substitutes. Materials and Method: Normal and osteoporotic bone strips were washed with jet lavage, cleaned of soft tissue, pasteurized, soaked in ethanol, freeze-dried and sterilized with 25 kGy of gamma radiation. For the spectroscopic studies bone samples at different stages of processing were cut cylindrically to fit into a Zirconium rotor. The molecular level structural arrangement at the interface of the collagen protein and hydroxyapatite interface in the bone samples was studied using SEM, SEM-Mapping and TGA. Water and other hydrogen/proton containing molecules were studied to see how they are engaged in the hydroxyapatite matrix. Results: In the proton 1D spectra, two peaks were observed, one at 5 ppm which corresponds to water and the other at 1 ppm, representing OH-/lipid protons. The relative peak height changed in different processed samples. Relative intensity of H20/Lipid decreased with different

processing steps especially with alcohol treatment and complete processing. In all the individual stages of processing the water to OH-/lipid ratio was imbalanced but with complete processing the intactness of water as well as the ratio improved. In Normal Bone, the H2O lines were

broader indicating that water is still bound with the inorganic matrix. In oesteoporotic cortical bone the water line was quite sharp indicating freely tumbling water. In normal cancellous bone, the water signal was low indicating significant reduction in water content. Conclusion: The present study explained the role of water and the inorganic matrix in the bone structure. The correlation of inorganic matrix with collagen helped in understanding the stabilizing structural properties of bone. Mimicking such interaction will help in synthesizing bone implant scaffold materials with the desired properties. The results indicated that the processing protocols used preserve the integrity of the bone sample.

193

P-031 – Biomechanical properties of cancellous bone cylinders after sterilization: a comparison of three different sterilization techniques S. Jung,1 A. Ignatius,2 A. Pruss,3 L. Dürselen,2 H. Reichel4 1 Department for trauma, reconstructive and orthopedic surgery, Hospital Weissenhorn, Germany 2 Institute of OrthopaedicResearch and Biomechanics University of Ulm, Germany

3 University Tissue Bank, Institute of Transfusion Medicine, Charité – Universitätsmedizin, Berlin, Germany

4 Orthopedic University Clinic Ulm, Germany

Introduction: In Germany, desinfection of human bone grafts is usually performed in three different ways: First, the Tutoplast- process and y-irradiation (TP), secondly, the peracetic acid treatment and following lyophilization (PA), third, the thermodesinfection and cryoconservation (TD). Every method is authorized by the German government. This biomechanical and μCT-study analyses the effect of the three different treatments on percent bone mass, bone density and trabecular thickness and the differences in compression stability compared to cryoconservated control groups. Method: We performed a symmetric preparation of cancellous bone cylinders out of vertebral bodies of calves. After shortening to 12 mm, every cylinder was analysed in a SKYSCAN-μCT by a definition of 12 μm. Focal points were percent bone mass (BV/TV), bone density (BD) and trabecular thickness (TT). Cylinders were paired randomized to the different cohorts. One cylinder of each pair was cryoconservated for three month at -80°C, the other was treated by one of the described methods. After 12 weeks, the μCT-scan was repeated after a two hour rehydration in ringer-lactate solution. After producing PMMA-feet for each cylinder, compression testings were performed. Results: Six lumbar spines were prepared, 36 pairs of cancellous bone cylinders were taken, followed by a randomized distribution. μCT-data showed following changes, indicated in percental changes before/after. In the TP-group there was an increase of BD 1,88%/-0,37% (treated/control), a decrease of BV/TV -0,17%/ 19,607% and an increase of TT 11,72%/ 9,59%. The thermodesinfected cancellous bone showed an increase of BD 17,48%/ 15,4% (treated/control), of BV/TV 5,24%/ 4,3% (treated/control) and of TT 8,57%/ 13,97. The PA-specimens showed an increase of BD 10,47%/ 3,76% (treated/control), of BV/TV 1,75%/ 1,58% and of TT 7,77%/ 16,6%. The biomechanic testings showed following results (changes in percent compared to the control groups): the modulus of elasticity after PA-treatment showed no changes, after the Tutoplast treatment a decrease of - 33,87% and after thermodesinfection of -18,89%. Elongation at fracture was decreasing in the PA- (-7,04%) and in the TP-group (-33,46%), but no changes after thermodesinfection. Maximum force showed the greatest effect in the TP-group (-52,52%), but also a negative effect after PA-treatment (-12,57%) and TD (-21,53%). Conclusion: The present study confirms different effects of chemical and thermic desinfection methods on compression stability of cancellous bone cylinders, but no changes in μCT datas. Greatest lost of stability was detected after the Tutoplast-process.

194

P-032 – Comparison of two different methods for the microbiological analysis of heart valves and processing liquids E. Scally,1 S. Shaw,1 L. Giurgola,2 C. Gatto,2 J. D’Amato Tothova2 1 Irish Heart Valve Bank, Irish Blood Transfusion Service, Dublin, Ireland 2 R&D Department, Al.Chi.Mi.A. S.r.l., Ponte San Nicolò, Italy

Introduction: Antibiotic solutions are used to decontaminate tissue allografts, however these solutions may induce bacteriostasis of microorganisms, resulting in false negative results. ResEP tube is a novel microbiological test with the ability to remove residual antibiotics from tissue and liquid samples. The aim of this study was to compare the standard microbiological testing used at the Irish Heart Valve Bank (IHVB) with the novel ResEP tube method. Methods: Eight heart valves were dissected and placed in an antibiotic cocktail containing gentamicin, metronidazole and flucloxicilline. The valves were then rinsed in RPMI1690 and cryopreserved in 10% DMSO. Tissue samples (1 cm3) obtained before and after decontamination were tested using Brain Heart Infusion, Thioglycollate and Sabouraud broths (Biomerieux) for 9 days. The antibiotic cocktail and rinse solutions were tested using the BacT Alert system (Biomerieux). The same samples were tested with ResEP tube in duplicate by IHVB and AL.CHI.MI.A, where direct inoculation in Tryptone Soy and Thiglycollate broths was also performed. Turbidity was monitored for 14 days and the isolated microorganisms identified. Results: Tissue samples prior to decontamination showed turbidity after 5 - 6 days using the IHVB method and after 24 – 48 hours with ResEP tube. Post decontamination, tissue and liquid samples remained negative with IHVB methods. One out of eight tissues was positive with the direct inoculation method. Six out of eight antibiotic cocktails and tissues were positive with ResEP tubes within 24h - 48 hours. The contaminants identified were of Bacillus and Staphylococcus species. Rinse fluids remained negative using both techniques. Conclusions: ResEP tube was the most sensitive and rapid method for the detection of contaminants for both tissue and liquids. These results highlight the importance of using an antibiotic neutralization system such as the ResEP tube.

195

P-033 – Living Donor, procurement up processing, following, ISO 9001 and quality control D. Pinto de Carvalho, S. Cuppens, P. Ferreira, E. Gerday, A. Marais, D. Dufrane Cliniques Universitaires Saint-Luc, Brussels, Belgium

Introduction: Femoral heads procurement of living donor is the first source material for production of securized bone grafts. Goal: The aim of this work studied the control way of bone tissue procurement up to processing and delivering on 2 consequent years (2011-2012) following an ISO9001-2008 quality system. Material/Method: Hospital institutions (orthopedics) working in partnership with our Unit of Tissue and Cell Therapy, proceed to procurement of femoral heads following a surgical intervention by a total hip prosthesis/or a fracture of femoral neck. After procurement, femoral heads are stored -80°C. For each living donor: screenings of donor’s history, informed consent, serological and bacteriological test are performed. Medical validation is then performed before tissue securization. After chemical and physical treatments, bone grafts are packed and sent to irradiation. An additional microbiological testing is then performed after gamma irradiation. Results: In 2011 and 2012, a mean of 2329 femoral heads were introduced to process 1165 and to exclude 987 tissues. Among securized tissues, different categories of grafts were produced: 65% of freeze-dried grafts (57% of freeze-dried bone sponge, 10% of freeze-dried cortico-sponge block, 28% of freeze-dried fragmented bone and 5% of 1⁄2 freeze-dried securized femoral head) and 35% of frozen grafts (65% of 1⁄2 frozen femoral heads, 18% of frozen 1⁄4 femoral head and 25% of frozen fragmented tissues. The major causes of tissues exclusion remains: (i) in 2011: 1126 femoral head excluded: 38% criteria of exclusion, 25% serological/bacteriological test unrealized, 17% serological/bacteriological incomplete tests, 15% others (no record, informed consent not completed) and 5% serological/bacteriological test positive; (ii) in 2012: 848 femoral head excluded: 32% criteria of exclusion, 20% serological/bacteriological test unrealized, 24% serological/bacteriological incomplete tests, 16% others and 8% serological/bacteriological test positive. Conclusion: The complete control of tissue obtention and processing can significantly reduce the exclusion rate by 10% between 2011 and 2012 following (i) education of collaborative institutions (for exclusion criteria) and (ii) direct control on serological-microbiological testing to avoid massive lost of the main bone source for securization.

196

P-034 – Precut DSAEK, experiences by the Euro Cornea Bank, Beverwijk P. Steijger, Y. Schuchard, W. F. van Marion Euro Cornea Bank, Beverwijk, The Netherlands

In 2012 the ECB decided to offer eye surgeons precut DSAEK. After research, the Gebauer SLc microkeratome was purchased as this system is fully automated and with the additional benefit of producing a posterior lamellar of 100 µm or less with a ‘Single Pass’ by use of DSAEK heads, ranging from 300 µm up to 550 µm. The quality of precut DSAEK has been established in collaboration with the ophthalmology department of the MUMC. Results have been published by Mr. Mor Dickman. During the training period in which we cut 200 corneas, obstacles were observed that needed to be resolved. 1. Liquid to maintain pressure in AAC 2. Applanation of the cornea on AAC 3. Removal of cornea from the AAC Recommendations for optimum results: Ad1. The PBS negatively affected the quality of the endothelium; therefore, it was decided to use MEM preservation liquid which showed significant improvement. Ad2. With the Gebauer SLc microkeratome a unique glass applanator is supplied making it possible to pre-determine graft diameter prior to cut. With this glass applanator an applanation of 9 - 10 mm resulted in the best outcome: a graft of >8 mm. In the beginning 9 mm was not always achievable but after slight modifications of the AAC this problem was quickly resolved by Gebauer. Ad3. The way in which the cornea is removed from the AAC strongly affects the endothelium quality. The anterior part must be placed back on the posterior part in order to assure that the shape of the cornea remains unchanged which is important for the endothelium. When the inner ring is removed from the socket, the cornea collapses. The cornea partly adheres to the inner ring resulting in preservation folds with dead cells, or the cornea completely adheres to the inner ring resulting in concentric folds with dead cells due to decrease of liquid pressure. In our opinion, the cornea must be kept in place on the socket while removing the inner ring. The cornea bank of Venice, Italy advised us to use ‘blue moons’ between the cornea and the inner ring. We have adopted this method. Meanwhile Gebauer is working on a definitive solution to this collapsing problem.

197

P-035 – Preparation of sternal bone for transplantation purposes for treatment in cardiosurgery J. Karkoska,1 M. Kalab,2 V. Lonsky2 1 National Cell and Tissue Center, Brno, Czech Republic 2 Department of Cardiac Surgery, University Hospital and Faculty of Medicine, Palacky University,

Olomouc, Czech Republic

Introduction: Authors describe development and preparation suitable bone graft for chest reconstruction after cardiothoracic operations. Goal: It seemed that sternal bone is an appropriate choice how to solve sternal dehiscence with poststernotomy defects. Preparation of sternal bone has to comply with Czech legislation Act on human tissues and cells 296/2008 Coll. and Decree 422/2008 Coll. in order to be used for transplantation purposes. Methods: Procurement of bone tissue is carried out at cooperating pathologic and forensic departments. Sternal bone is procured before autopsy procedure. It is removed after surgical preparation of the donor's body. The tissue is aseptically packed and marked with a unique identification number. Procurement and processing is subject to the quality control - negative results serology, microbiology and suitable autopsy results, etc). The processing then takes place in clean rooms in the special isolator technology (class A cleanness, background class C) that is used for processing musculoskeletal tissues. Sternal bone is formed into a basic structure with manubrium and sternal body and treated by decontamination solutions. Graft is stored at −80°C. Results: Amount of sternal bone transplants is limited because the tissue has to be procured from the donor s body before autopsy and sometimes it is destructed by emergency ambulance in urgent life-saving cases. We prepared and used 6 grafts for transplantations at this time. Conclusions: Close cooperation between Tissue establishment and clinicians led us into development of unique solution how to use bone graft in cardiosurgery.

198

P-036 – Ultra-thin lamellae in one step using Gebauer SLc Microkeratome System E. Agustí, N. Otero, E. M. Martínez-Conesa, M. L. Pérez, A. Vilarrodona, E. Trias Transplant Services Foundation (T S F), Barcelona, Spain

Introduction: During the period from January 2012 to July 2013, 102 donor corneas were cut with Gebauer SLc Microkeratome by a technician in our tissue bank, usually the one day prior to the lamellar keratoplasty surgery. From February 2013 onwards a new deeper cutting heads was introduced to the cutting procedure, providing thinner precut tissues for endothelial keratoplasty. Goal: To present the results, since TSF started using the new deeper cutting heads with 41 corneas cut and sent to 11 implanting centres for endothelial keratoplasty (DSAEK) Methods: 41 corneas from February to July 2013 were selected to be sectioned under GMP conditions in our tissue bank using semiautomatic Gebauer SLc microkeratome with different depth DSAEK heads paying special attention to the mentioned new heads. Central corneal thickness before and after microkeratome sectioning was measured by ultrasonic pachymetry. Results: From 41 corneas, 2 of them were dissected for anterior lamellar keratoplasty (ALK) and 39 for DSAEK. Taking into account the corneas for DSAEK, the average donor age was 58,8 years old and the mean endothelial cell density was 2744.6 cells/mm2. The initial and final mean corneal thicknesses were 604.9 and 96,2 mm, respectively. The final lenticule thickness was not measured in 7 cases, in those cases an estimate (initial measurement-cutting head size=Final measurement) was done. In 1 case, 400mm depth head was used, in 6 cases 450mm, in 11 cases 500mm, in 13 cases 550mm and finally in 7 cases 600mm depth heads were used. In 1 cornea we had a posterior perforation or “button hole”. The operator was the same person in 32 out of 40 cases (80%). In relation to the first post-operative control, 90% of the transplants had a satisfactory evolution and the surgeons reported some difficulties in handling the lamellae in 2 cases. Conclusion: Gebauer SLc microkeratome is a reliable system to acquire ultra-thin endothelial lamellae in one step in a Tissue Bank, fulfilling the increasing demand of thinner pre-cut tissues for substituting just endothelial layer of the cornea of each patient and facilitating the surgeon's work.

199

P-037 – Storage and qualification of viable in toto human amniotic allograft: technology transfer to a tissue bank and outcome for bone repair F. Gindraux,1,2,3 R. Laurent,2,4 A. Nallet,5 P. Layrolle,6 L. Nicod,2 L. Obert1,2 1 Orthopaedic and Traumatology Surgery Service, University Hospital of Besancon, France 2 Intervention, Innovation, Imagery, Engineering in Health (EA 4268), SFR FED 4234, University of Franche-

Comté, Besancon, France 3 Clinical Investigation Centre in Biotherapy, University Hospital of Besancon, France

4 Paediatric Surgery Service, University Hospital of Besancon, France

5 Novotec, Lyon, France

6 Inserm U957, Laboratory Physiopathology of Bone Resorption, Faculty of Medicine, University of Nantes, France

Introduction: Human Amniotic Membrane (hAM) has good potential to enhance tissue regeneration thanks to its properties as a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. To investigate potential of hAM as an advanced therapeutic medicinal product for bone repair, we aimed to assess the influence of published tissue culture and/or cryopreservation methods on cell viability and tissue structure; and secondly to confirm its in vitro osteogenic potential. Goal: We aim to implement these procedures in a tissue bank, i.e. easy processes to store hAM containing viable cells and simple and rapid qualification methods to verify quality and phenotype of grafts before release for use. Methods: We tested different published culture media (in osteogenic condition or not) and cryopreservation storage conditions. Cell viability, tissue structure and phenotype were evaluated respectively by trypan blue, Giemsa and Von kossa stainings and results were compared to histological analysis. Results: There was no significant decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data showed that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Second, hAM could be osteodifferentiated in vitro by an osteogenic medium and that the culture involves structural alterations of epithelium related to modified cell function, especially in this condition. Conclusion: For tissue engineering outcomes, we suggest storing hAM with as little manipulation as possible, to preserve cell function and tissue structure. We report simple and rapid processes that can easily be implemented in a tissue bank to qualify the allograft.

200

P-038 – Transplantation of in vitro cultured autologous keratinocytes and fibroblasts suspended in Platelet Leukocyte Rich Gel with growth factors in the treatment of burns W. Łabuś,1 M. Kawecki,1,2 A. Klama-Baryła,1 D. Hoff-Lenczewska,1 M. Kraut,1 M. Maj,1 J. Glik,1,2 M. Nowak1 1 Centre for Burn Treatment in Siemianowice Śląskie, Poland

2 Department of Health Science, University of Technology and Humanities in Bielsko Biała, Poland

Introduction: Treatment of extensive burn wounds with a deficit of donor sites requires to implement alternative methods of treatment. In 2008 a transplantation of in vitro cultured autologous keratinocytes and fibroblasts suspended in leukocyte platelet rich gel PLRG with growth factors was introduced into clinical practice in the Centre For Burn Treatment in Siemianowice Slaskie (Poland). The in vitro culture of autologous keratinocytes and fibroblasts allows to obtain the appropriate number of cells, sufficient to cover burn wounds. Goal: Evaluation of the healing of burn wounds after transplantation of in vitro cultured autologous keratinocytes and fibroblasts suspended in leukocyte platelet rich gel PLRG with growth factors. Methods: In the period 2008 to 2013 was performed 90 autologous keratinocytes grafts for 58 recipients and 25 autologous fibroblasts grafts for 22 recipients. The cells were suspended in leukocyte platelet rich gel PLRG with growth factors. Result: After autologous keratinocytes and fibroblasts transplantation much faster wound closure was observed. A histological examination in the fifth day after transplantation depicted the elements of new epidermis. In the tenth day after transplantation a keratinizing layer of the epidermis was observed in the histological images. Conclusion: The usage of in vitro cultured autologous keratinocytes and fibroblasts suspended in leukocyte platelet rich gel PLRG with growth factors leads to fast and stable closure of burn wound.

201

P-039 – Amnion as a potential source of stem cells for wounds treatment D. Hoff-Lenczewska, M. Kawecki, A. Klama-Baryła, W. Łabuś, J. Glik, M. Kraut, M. Nowak

Burn Treatment Centre, Siemianowice Śląskie, Poland

Introduction: Amnion transplants gives promising results of wound healing, with minimal inflammation and scar formation; which suggest that amnion membrane has the similar features as fetal tissue. Amnion supports wound healing and regeneration of tissue. Most interesting are amnion mesenchymal stem cells because of their ability to differentiate and possibility of usage of therapeutic potential in regenerative medicine. Ability of transdyfferentiation into various tissues has been already proved. Another important feature of these cells are immunomodulative properties. Goal: Aim of this study was to check of the potential of amnion as a source of noninvasive and easily accessible source of stem cells achieved by biotechnological methods with the implementative potential of wounds treatment. Methods: In order to choose the optimal method of cell isolation following enzymes were used: dispase, trypsin, collagenase I. In addition, isolation by homogenization was performed to compare with enzymatic isolation. We compared four culture media. To assess cell migration during wound healing in vivo Wound Healing Assay was used. This assay demonstrated that amniotic cells also affects the cells of the dermis. Result: Method of isolation and culture of amnion cells was selected on the basis of statistically significant results on cell viability and apoptosis. The influence of amnion cells on fibroblasts resulting in faster wound closure time in an in vitro assay. Conclusion:

1. Amnion is a cheap and easily available material for the isolation of cells, including stem cells. 2. There are significant differences in the number and viability of cells obtained according to the method of isolation and culture conditions. 3. Amnion cells affect the ability of fibroblasts to closure of the wound.

202

P-040 – GMP-compliant isolation and expansion of human amniotic mesenchymal stem cells with human alternatives to fetal bovine serum E. Lugmayr,1,2 D. Theiß,1,2 K. Witzeneder,1,2 A. Lindenmair,2,3 C. Gabriel,1,2 S. Hennerbichler-

Lugscheider1,2

1 Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria 2 Austrian Cluster for Tissue Regeneration 3 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Linz/Vienna, Austria

Human amniotic membrane represents a rich source of stem cells to be used in regenerative medicine and tissue engineering. Human amniotic mesenchymal stem cells (hAMSC) may act as an alternative to bone marrow derived stem cells in future as collection is easier and non invasive and can be isolated from human amniotic membrane by enzymatic digestion using a collagenase solution containing fetal bovine serum (FBS). For use in regenerative medicine and tissue engineering expansion of the cells is necessary because of the high amount of cells needed. So far, expansion protocols require the addition of FBS though batch to batch variations are known. Furthermore, the risk of xeno-immunization in patients and the potential incorporation of xenogenic factors make it unsuitable for clinical applications. Therefore protocols using human alternatives according to GMP requirements should be defined. After informed consent seven human term placentae were obtained from Caesarean section. Amniotic membrane was removed from chorion and incubated for two hours in a collagenase solution supplemented with or without FBS. The cells were expanded for three passages in media supplemented either with 5% human platelet lysate (hPL) or 10% human serum (huS). At a confluence of at least 80% the cells were subcultured and population doubling times were determined. No differences in cell counts and morphology were observed that could be attributed to the presence of FBS in the collagenase solution for isolation. Furthermore we compared collagenase (used to date, for research only) with a GMP-compliant collagenase and obtained similar results. Confluence was already reached after three to four days independent of the added supplement whereas differences in morphology between cells expanded with hPL and huS were noticed. In summary, we were able to establish a GMP-compliant protocol for the isolation of hAMSC without the need of FBS in the collagenase solution as well as the equal applicability of GMP-grade collagenase. Regarding cell expansion it is possible to substitute FBS by either huS or hPL.

203

P-041 – Synthesis of cartilage-like neo-tissue by the culture of human stem cells over collagen and heparan sulfate scaffolds C. Sanjurjo-Rodriguez,1,2 E. Rendal-Vázquez,2 A. H. Martínez-Sanchez,1,2 T. Hermida-Gómez,1,2 I. M. Fuentes-Boquete,1,4 J. Buján,5 F. J. De Toro,1,3 F. J. Blanco,1,2 S. Díaz-Prado1,3 1 INIBIC, A Coruña, Spain 2 CHUAC, A Coruña, Spain 3 Medicine Department, CHUAC, A Coruña, Spain 4 Medicine Department Universidade de A Coruña, A Coruña, Spain 5 Medical Specialties Department, Universidad de Alcala de Henares, Madrid, Spain

Introduction: Cartilage Engineering may provide a promising alternative solution to current methods of cartilage repair. The aim of this study was to evaluate chondrogenesis of human Bone Marrow Mesenchymal Stem Cells (hBM- MSCs) cultured on type I collagen (Col I) and different concentrations of heparan sulfate (HS) scaffolds. We also studied the quality of the neo-engineered cartilage-like tissue. Materials and Methods: hBM-MSCs were cultured over Col I and HS scaffolds, with two different culture mediums: chondrogenic differentiation medium supplemented with Transforming growth factor β-3 (TGFβ-3) and DMEM with 20% of fetal bovine serum (control cultures), both of them supplemented with Parathyroid Hormone related-Protein (PTHrP). Chondrogenesis and the neo-synthesized tissue were evaluated through histochemical, immunohistochemical, electron microscopy (EM) and molecular biology analysis. Col released to the supernatants by the constructs was also measured by Elisa assays. Results: hBM-MSCs grown throughout all the tested scaffolds, showing the stimulated ones the higher percentage of cells (more than 80%), related to the whole scaffold area. Also, proliferation was higher in chondrogenic medium than in controls. Differentiated constructs extracellular matrix (ECM) showed type II Col (Col II), aggrecan (Agg) and proteoglycan positivity. Furthermore, von Kossa staining showed a more calcified ECM in controls than in chondrogenic cultures. On the other hand, expression of characteristic chondrocytic genes was detected. Although we did not find differences in COL II relative expression levels (R.E.L.) (p-value>0.05), we found differences in SOX9, which showed higher R.E.L. in chondrogenesis than in controls (p-value<0.05). Also, we found differences in AGG R.E.L., being higher in differentiated Col I+3%HS (p-value<0.05). EM confirmed the presence of rounded cells and ECM in chondrogenic cultures. Finally, Col released was detected only in differentiated culture supernatants. Conclusions: Data indicated that Col I and HS scaffolds were useful to allow the hBM-MSCs growth and differentiation, in chondrogenic medium. Moreover, scaffolds favor the differentiated phenotype maintenance and the synthesis of a new tissue that could be useful in cartilage engineering. Acknowledgments: B. Parma (OPOCRIN S.P.A.); CAM (S2009/MAT-1472); CIBER-BBN CB06-01-0040; P. Esbrit (Fundación Jiménez Díaz); REDICENT; Universidade de A Coruña (UDC); SAI-UDC; CSR is beneficiary of a Diputación de A Coruña fellowship.

204

P-042 – Cartilage tissue engineering using human mesenchymal stem cells cultured on collagen biomaterials C. Sanjurjo-Rodriguez,1,2 E. Rendal-Vázquez,2 A. H. Martínez-Sanchez,1,2 T. Hermida-Gómez,1,2 I. M. Fuentes-Boquete,1,4 J. Buján,5 F. J. De Toro,1,3 F. J. Blanco,1,2 S. Díaz-Prado1,3 1 INIBIC, A Coruña, Spain 2 CHUAC, A Coruña, Spain 3 Medicine Department, CHUAC, A Coruña, Spain 4 Medicine Department Universidade de A Coruña, A Coruña, Spain 5 Medical Specialties Department, Universidad de Alcala de Henares, Madrid, Spain

Introduction: Promising methods for cartilage repair are based on engineer a cartilage-like tissue, culturing human mesenchymal stem cells derived from bone marrow (hBM-MSCs) over biodegradable scaffolds. The aim of this study was to evaluate the hBM-MSCs chondrogenesis when cultured on collagen (Col) biomaterials and their potential usefulness in Cartilage Tissue Engineering. Materials and Methods: hBM-MSCs were cultured in chondrogenic medium supplemented (Lonza, Belgium) with Transforming growth factor β-3 (TGFβ-3) and in DMEM (Lonza) with 20% of fetal bovine serum (control), over 4 different Col biomaterials: Col I + Col II (C1C2); Col I + Col II + heparan sulfate (C1C2HS); Col I + Col II + chondroitin sulfate (C1C2CHS) and Col I + heparine (C1OLH3). Chondrogenic differentiation was tested through immunohistochemical and histochemical analysis. Constructs were also evaluated by means of electron microscopy and molecular biology. Also, Col concentration was determined in the supernatant cultures, by Elisa assays. Results: hBM-MSCs were capable to grow in the surface and inside all the biomaterials. The percentage of cells regarding the total scaffold area was higher than 75% in C1C2HS and C1C2CHS, and higher than 50% in the remaining scaffolds. Proliferating cell nuclear antigen (PCNA) immunostaining showed cellular proliferation in all the scaffolds, obtaining the highest values in C1C2 and C1C2HS. By electron microscopy, we detected a high cell secretory activity, big amount of mitochondria, oval/rounded cell morphology, cell attachment to the biomaterial and extracellular matrix (ECM). This ECM was positive for Col II and proteoglycan, in all the biomaterials except in C1OLH3. Regarding to relative expression levels (REL) of characteristic cartilage genes, significant differences were found between all biomaterials in SOX9 and COL II (p-values 0.05). Moreover, Col was detected in culture supernatants. Conclusions: The different evaluated Col biomaterials were useful for the hBM-MSCs growth. These cells were also capable to differentiate over C1C2HS and C1C2CHS, to chondrocyte-like cells. These biomaterials could be appropriate to Engineer Cartilage. Acknowledgments: B. Parma (OPOCRIN S.P.A.); CAM (S2009/MAT-1472); CIBER-BBN CB06-01-0040; REDICENT; Universidade de A Coruña (UDC); SAI-UDC; CSR is beneficiary of a Diputación de A Coruña fellowship.

205

P-043 – Skin equivalent with human amniotic mesenchymal cells and human amniotic epithelial cells on an amniotic membrane scaffold

A. Sanluis Verdes,1 E. Rendal-Vázquez,1 N. Sanluis Verdes,2 N. Doménech García,3 M. T. Yebra-Pimentel Vilar,4 M. Fraga Mariño,5,6 J. Sánchez Ibáñez1 1 Criobiology Unit – Tissue Bank, CHUAC, A Coruña, Spain 2 General and Digestive Surgery Department, CHUAC, A Coruña, Spain 3 Biobank, CHUAC, A Coruña, Spain 4 Anatomopathologic Department, CHUAC, A Coruña, Spain 5 INIBIC, A Coruña, Spain

6 CHUAC, A Coruña, Spain

Introduction: Regenerative medicine is based on the use of stem cells to generate biological substitutes and improve tissue functions, restoring damaged tissue. For this to take place we need stem cells, a support and growth and differentiation factors. All of which can be obtained from Human Amniotic Membrane (HAM). Goal: The aim of this study was to develop a skin equivalent with Human Amniotic Mesenchymal Cells (hAMCs) and Human Amniotic Epithelial Cells (hAECs) on a HAM scaffold. Methods: Both hAMCs and hAECs we extracted from five HAM after obtaining informed consent from the donors and after a double digestion with collagenase and trypsin-EDTA respectively and they were characterized through flow cytometry for mesenchymal stem markers (CD44, CD73 and CD105) and by multipotential analysis towards the mesodermic lineage. The hAMCs were seeded on a culture dish and after 24 h on top of these a de- epithelialized HAM (with trypsine/EDTA 1%, 30 min at 37°C) and later the hAECs were seeded over these and were cultured using the submerged method for a week. This skin equivalent underwent a histological (hematoxylin-eosin (H-E), Masson’s trichrome, alcian blue and Safranin O), immunohistochemic (cytokeratin A1/A3, K14, K10, vimentin and p63) and inmunofluorescence (laminin) studies after culture. Result: Both the hAMCS as well as the hAECS extracted from the HAM presented markers for mesenchymal stem cells on their surface and also the capacity of differentiation of the mesodermic lineage (adipo-, osteo- and chondrocytes). The results of skin equivalent (H-E) presented a monostratified epidermis formed by cytoqueratin positive hAECs on a basement membrane. The basement membrane was laminin positive. The dermis consisted in vimentin positive hAMCs in a collagen matrix (Masson’s trichrome positive), mucopolysaccharides (alcian blue positive) and proteoglycans (Safranin O positive). The epidermis was made up of a basal strata formed by epithelial stem cell (P63 y K14 positives) but it does not have suprabasal strata (K10 negative). Conclusion: Our findings suggest that skin equivalent has good application prospects in regenerative medicine.

206

P-044 – Dermal equivalent with stem cells A. Sanluis Verdes,1 E. Rendal-Vázquez,1 N. Sanluis Verdes,2 N. Doménech García,3 M. Fraga Mariño,4,5 M. T. Yebra-Pimentel Vilar,6 J. Sánchez Ibáñez1 1 Criobiology Unit – Tissue Bank, CHUAC, A Coruña, Spain

2 General and Digestive Surgery Department, CHUAC, A Coruña, Spain

3 Biobank, CHUAC, A Coruña, Spain

4 INIBIC, A Coruña, Spain

5 CHUAC, A Coruña, Spain 6 Anatomopathologic Department, CHUAC, A Coruña, Spain

Introduction: Skin equivalents have been developed in order to reproduce in vivo conditions. A dermal layer provided better outcomes such as higher skin graft uptake rate, quicker healing process, better cosmetic features and skin pliability, mechanical resistance and satisfactory cosmetic results. Several materials have been applied as a dermal equivalent to develop more practical skin equivalents. However, the previous dermal and skin equivalents have some drawbacks because they contained exogenous materials in the dermal equivalents, such as bovine collagen, human allogenic dead dermis, synthetic polymers, and so on, and these substances are expensive, difficult to obtain and are a source of infections. Goal: The aim of this study was to develop an in vitro dermal equivalent synthesized from the culture alone without exogenous materials from human Amniotic Mesenchymal Cells (hAMCs) or from human umbilical cord Wharton’s jelly mesenchymal Stem Cells (hWJSCs) for three weeks. Methods: The postconfulent cultures of hAMCs and hWJSCs were cultured for three weeks in a 3:1 mixture of DMEM/Ham-F12 supplemented with 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (0.4 μl/ml), triiode-thyronine (1.3 ng/ml), adenine (24 μl/ml), transferrin (5 μg/ml), L-ascorbic acid-2-phosphate (50 μg/ml) and 1% penicillin–streptomycin until a fibrous sheet was produced. On this dermal equivalent histological (hematoxylin-eosin (H-E), Masson’s trichrome, alcian blue and Safranin O) and immunohistochemic (vimentin, collagen type I and fibronectin) studies were carried out after culture to check for the main components of an extracellular matrix. Result: Histologically (H-E), the fibrous sheet showed dermis-like tissue that consisted of an extracellular matrix formed by hAMC or hWJSCs (vimentin positive) and revealed components of dermal matrix such as collagen fibers (Masson-trichrome staining and type I collagen positives), mucopolysaccharides (alcian blue positive), proteoglycans (Safranin O positive) and fibronectin (fibronectin positive) which were diffusely expressed. Conclusion: These findings demonstrated that a dermal in vitro equivalent with hAMCs and hWJSCs can be synthesized to serve as a scaffold to synthesize a skin equivalent that could be used for in vivo grafting.

207

P-045 – Endothelialized dermal equivalent with human umbilical cord Wharton’s jelly mesenchymal stem cells A. Sanluis Verdes,1 E. Rendal-Vázquez,1 N. Sanluis Verdes,2 N. Doménech García,3 M. T. Yebra-Pimentel Vilar,4 M. Fraga Mariño,5,6 J. Sánchez Ibáñez1 1 Criobiology Unit – Tissue Bank, CHUAC, A Coruña, Spain 2 General and Digestive Surgery Department, CHUAC, A Coruña, Spain 3 Biobank, CHUAC, A Coruña, Spain 4 Anatomopathologic Department, CHUAC, A Coruña, Spain 5 INIBIC, A Coruña, Spain

6 CHUAC, A Coruña, Spain

Introduction: One of the major problems of tissue-engineered skin is its failure in the vascularization as they are colonized by neoformed blood capillaries, and the blood flow appears 10-15 days after the graft has been implanted making them incompatible with the survival of the graft. When a skin equivalent is grafted with endothelial cells, tubular structures can be observed 4 days after grafting. The human umbilical cord Wharton’s jelly Mesenchymal Stem Cells (hWJSCs) could be a possible source for cells with the ability to differentiate into fibroblasts and endothelial cells in vitro. Goal: Investigate the potential of hWJSCs as a possible source of cells to induce the differentiation towards cells similar to fibroblasts and endothelial cells and the potential of human Amniotic Membrane (HAM) as a possible scaffold for the growth of these differentiated cells. Methods: The hWJSCs were extracted using the explanted method from three human umbilical cords, after informed consent from the donors and were cultured in a medium for induction to fibroblasts (DMEM, 5% SBF, 5 ng/ml, TGF-b, 5 ng/ml IGF-II, 1 μg/ml EGF, 1 μg/ml insulin, 1 ng/ml bFGF, 2.5 mg/L Asc-2-P and 1% P/S) and endothelial cells (DMEM, 50 ng/ml VEGF, 2% SBF and 10ng/ml bFGF) for 21 days. The differentiated hWJSCs were seeded on the stromal face of the HAM for a week. The differentiation was confirmed through the expression of markers such as vimentin for the fibroblasts and CD31 for the endothelial cells and the morphology was observed through hematoxylin-eosin (H-E) and Masson’s trichrome staining. Result: Histologically (H-E), it could be observed how the hWJSCs differentiated to fibroblasts and endothelial cells which adhered to the stromal face of the HAM. An extracellular matrix was synthesized (Masson’s trichrome positive) where it was possible to differentiate vimentin positive cells (fibroblasts) and CD31 positive cells (endothelial cells). Conclusion: These findings demonstrated that it is possible to synthesize an endothelialized dermal equivalent in vitro from hWJSCs on a HAM scaffold that could be used for the reconstruction of a skin equivalent.

208

P-046 – Use of umbilical cord as a source for cells and amniotic membrane as a scaffold for the treatment of neurodegenerative diseases A. Sanluis Verdes,1 N. Sanluis Verdes,2 E. Rendal Vázquez1

1 Criobiology Unit –Tissue Bank, CHUAC, A Coruna, Spain 2 General and Digsetive Surgery Department, CHUAC, A Coruna, Spain

Introduction: The main source of cells for tissue engineering are nervous tissue Schwann cells and mesenchymal cells derived from bone marrow, but their clinical application is limited. Mesenchymal stem cells derived from human umbilical cord Wharton's jelly mesenchymal Stem Cells (hWJSCs) can be employed as a source of cells for regeneration of damaged neural tissue because of their accessibility, capability of differentiation and proliferation of the three cell lineages. To avoid loss of cells once injected and promote nerve regeneration human amniotic membrane (HAM) scaffold is used as a biomaterial with many advantages such as lack of immunogenicity. Goal: Investigate the potential of hWJSCs as a possible source of cells to induce their differentiation into dopaminergic cells and the potential of HAM as a possible scaffold for the growth of these differentiated cells. Methods: The hWJSCs were extracted using the explanted method from ten human umbilical cords, after informed consent from the donor. The hWJSCs were characterized by flow cytometry for mesenchymal stem cell markers (CD44, CD73 and CD105) and cultured in neurogenic induction medium for 21 days. Differentiation was confirmed by the expression of neuronal (medium neurofilament and nerve growth factor receptor), glial (glial fibrillary acidic protein and S-100) neuronal or glial precursor (vimentin) and dopaminergic cells (tyrosine hydroxylase) markers. The HAM epithelium was removed with trypsin / EDTA (1%, 30 min at 37 ° C) and subsequently differentiated hWJSCs were seeded onto it. Result: The hWJSCs expressed mesenchymal stem cells markers. The morphological differentiation of hWJSCs into nerve cells was evident after 4-5 days when they acquired an elongated and multipolar shape. After 21 days, this differentiated cells expressed neuronal, glial, neuronal and glial progenitor and dopaminergic cells markers. The HAM epithelium was completely removed without affecting the components of the basement membrane or matrix, getting the differentiated hWJSCs to accede to it. Conclusion: The hWJSCs and HAM have great potential for use in regenerative medicine.

209

P-047 – Ciprofloxacin regulates negatively egr-1 transcriptional activity in human primary tenocytes F. Martinez-F.,1,2 C. Machuca-R.,3 K. Guzman-M.,1 A. Barrera-L.,1 F. A. Jimenez-O.,2 J. A. Madinaveitia-V.4 1 Skin & Tissue Bank at the National Institute of Rehabilitation, Mexico City, Mexico 2 Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City, Mexico 3 Molecular Therapeutic Lab, UMIEZ-FES Zaragoza, UNAM, Mexico City, Mexico 4 National Institute of Rehabilitation, Mexico City, Mexico

Introduction: Tenocytes proliferation rate is an essential factor for remodeling and maintenance of tendon integrity. Negative balance of the rhythm of cell proliferation is a key factor for inflammatory process and tendon rupture. Helicases inhibitors such as fluoroquinolones have been reported as factor for tendon rupture and currently used as antibiotic for tissues recovering. Hereby, we analyze the effect of ciprofloxacin on rate of cell proliferation in human primary tenocytes and the effect on the transcriptional regulation of egr-1 promoter based on transduction of adenoviral vector Adegr1-Luc. Materials and methods: Cells and adenoviral vectors: No replicative recombinant adenovirus (AdEgr1-Luc) were packaged at large scale in HEK-293 cells and purified according to the current protocol based on cesium chloride gradient protocol for in vivo application. Viral Stock was titled by plaque assay and OD. Human primary tenocytes (HPT) were obtained based on collagenase digestion protocol and cultured in DMEM/F12 Media supplemented with 10% HI-FBS and antibiotics under standard culture conditions for 1 week and stored for experimental procedure. 5x104 Tenocytes were seeded/well. After 12 hrs, cells were infected with AdEgr1-Luc at 50 MOI s during two hrs. in serum reduced media. After infection, cells were kept in 1% of FBS for 24 hours at environment standard conditions for culture and ciprofloxacin at 5 a 10 μg/ml (15, 30, 60, 90 and 120 seg). Protein extraction was performed at 2, 6 and 12 hrs based on Cell Glo Lysis Buffer (Promega corp.) and luciferase activity was quantified using a multidetector DTX-880. Results: Human tenocytes transduced with AdEgr1-Luc are positively responsive to (10%) of Egr-1 promoter (18,233 LC/s). Luminescent activity in presence of ciprofloxacin was observed at 2, 6 and 12 hrs (1,490 LC/S; 1,704.66 LC/s and 1,851.33 LC/s, respectively). Conclussion. Ciprofloxacin inhibits transcriptional activity of egr-1 promoter in human primary tenocytes. However, this fact is actually studies to determinate the signal transduction of regulation in tenocytes and the effect on cell proliferation rate by Cell proliferation assays. Acknowledgments: This research project is granted by the National Council of Science and Technology of México trough the project FOSIS/CONACYT-11-1-161624.

210

P-048 – Effect of ciprofloxacin on proliferating rate of human primary tenocytes and Fibroblasts H. Sandoval-Z.,1 J. A. Madinaveitia-V.,1 A. Barrera-L.,2 C. Machuca-R.,3 F. Martinez-F.4,5 1 National Institute of Rehabilitation, Mexico City, Mexico 2 Molecular Biotherapeutic Program, Skin & Tissue Bank at the National Institute of Rehabilitation, Mexico

City, Mexico 3 Molecular Therapeutic Lab, UMIEZ-FES Zaragoza, UNAM, Mexico City, Mexico

4 Skin & Tissue Bank at the National Institute of Rehabilitation, Mexico City, Mexico

5 Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City, Mexico

Introduction: Fibroblast cell proliferation rate is an essential factor for remodeling and maintenance of tissue integrity. Negative balance of the rhythm of cell proliferation is a key factor for inflammatory process and burns. Helicases inhibitors such as fluoroquinolones have been reported as factor for tendon rupture. Hereby, we analyze the effect of ciprofloxacin on rate of cell proliferation in human primary fibroblasts and tenocytes. Materials and methods: Cells. Human primary tenocytes (HPT) were obtained based on collagenase digestion protocol and cultured in DMEM/F12 Media supplemented with 10% HI-FBS and antibiotics under standard culture conditions for 1 week and stored for experimental procedure. 5x104 cells were seeded/well and kept in 10% of FBS for 24 hours at environment standard conditions for culture and ciprofloxacin at 5, 10, and 20 μg/ml. Crystal violet assay were performed at several days of culture (1-10 days) and proliferation rate were quantified by transmittance 520 nm in a multidetector DTX800 (Beckman coulter). Results: Fibroblasts shows a reduced pattern of growth depending of ciprofloxacin concentration. Tenocytes growth more slowly than fibroblasts (0.5 folds reduction compared to Fibroblasts). No effect of cell proliferation was observed at low concentration of ciprofloxacin (5, 10 μg/ml). Conclusion: Ciprofloxacin inhibits cell growth in human primary tenocytes and fibroblasts. However, this fact is actually studied to determinate the signal transduction of regulation in tenocytes and discern the effect on cell proliferation. Acknowledgments: This research project is granted by the National Council of Science and Technology of México. Grant FOSIS/CONACYT-Salud-2011-1-161624.

211

P-049 – Brain and tissue banking perspectives in Greece D. Anestakis,1,2 S. Petanidis,3 A. Salifoglou,3 M. Tsolaki4,5 1 Laboratory of General Biology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

2 Greek Association of Alzheimer’s Disease and Related Disorders, Thessaloniki, Greece 3 Department of Chemical Engineering, School of Engineering, Aristotle University of Thessaloniki, Thessaloniki, Greece 4 3rd Department of Neurology, Aristotle University of Thessaloniki, Thessaloniki, Greece 5 Panhellenic Federation of Alzheimer’s Disease, Thessaloniki, Greece

Introduction: In the last decade, research on human brain tissues increased our understanding of the nervous system function and mechanisms. Brain banks are vital in the scientific research of neurodegenerative disorders because they have the facilities and the expertise to preserve classify and distribute specimens for research, abiding by the local ethical and legal framework. Goal-Methods: Because most human neurodegenerative diseases are mainly or only observed in humans, the availability of specimens (i.e. tissues and body fluids) in brain banks and biobanks is a major resource for research. Brain banks have proved to be excellent platforms and vital facilities for clinical research. Recently, postmortem human brain tissues have contributed to the development of a genetic test for Huntington's disease (HD) and a treatment for Parkinson's disease (PD). In this regard, neurochemical and anatomical studies focusing on postmortem brain tissue today can provide details of a disease process and potentially its etiology. Concurrently, development of new molecular and neurobiological methods as well as new computer-assisted quantification techniques can assist brain tissue research. That collective prospect, in turn, leads to an increased demand for postmortem tissue in medical research and thus the need for the establishment of a brain bank. For a brain bank to be created, however, certain standardized and agreed upon criteria must be met, involving protocol-based tissue handling and collection of clinical data. Conclusion: The fundamental tenets of such an organized brain bank in North Greece are analyzed in the framework of (inter)national networks and placed in perspective so as to support its establishment that merits substantive contributions to multidisciplinary neurological disease research worldwide. This abstract focuses on current brain banking organization and management, as well as the likely future direction of the brain banking field.

212

P-050 – Effects of PRP and Platelet Lysate on stimulation of human tenocyte cultures T. Schmidt,1 F. Klatte-Schulz,1 B. Wildemann,1 S. Scheffler,2 A. Pruss3 1 Julius Wolff Institut, Charité- Universitätsmedizin, Berlin, Germany 2 Chirurgisch Orthopädischer Praxisverbund, Berlin, Germany

3 University Tissue Bank, Institute of Transfusion Medicine, Charité – Universitätsmedizin, Berlin, Germany

Introduction: Different preparations of platelet rich plasma (PRP) or platelet lysate (PL) are under investigation regarding their stimulating impact on tissue healing. This positive effect is attributed to the high content of growth factors which are known to trigger angiogenesis and tissue regeneration. The aim of this pilot study was to compare the effect of PL and PRP on human tenocytes regarding cell activity and gene expression in vitro. Material and methods: PL or PRP from two healthy donors was prepared following standard protocols or the instructions of the manufacturer. Tenocytes from 4 male and 4 female donors were isolated from supraspinatus tendon biopsies by collagenase digestion. A total of 1x104 cells were seeded into 24-well plates and an Alamar blue test was performed to analyze cell activity. Afterwards cells were stimulated with 10% PRP or 10% PL in cell culture medium with 2% FCS. After 5 days cell activity was again analyzed. RNA was isolated and gene expression analysis of collagen I, III and decorin was performed by Real-Time PCR. Results: Stimulation with PRP resulted in a significant higher increase of cell activity compared to the stimulation with PL. Collagen I expression was significantly decreased compared to negative control after stimulation with PRP. PL showed significantly increased collagen I expression compared to the respective PRPs. However, collagen I expression was decreased in all groups compared to positive control. Collagen III expression was significantly increased after stimulation with both PRP and PL compared to negative control. Therefore, collagen I to III ratio was significantly decreased in both PRP groups and female PL compared to negative control. Discussion: Tendon healing is limited due to poor vascularity and intrinsic healing capacity. This pilot study showed comparable increase of tendon cell activity after stimulation with PRP and PL. Collagen I/III ratio showed a shift to collagen III in both treatment groups. However, collagen I expression was significantly higher in PL stimulated cells compared to PRP stimulated cells. This could indicate a higher regenerative capacity of PL, because collagen I is the most important matrix protein in tendons and responsible for mechanical competence.

213

P-051 – Polymer carriers for thermal controlled manufacturing and detachment of cell sheets – ‘Polycell’ A. Klama-Baryła

Centrum Leczenia Oparzeń/Centre for Burns Treatment, Siemianowice Śląskie, Poland

Introduction: Based on previous research scientific consortium, has developed matrix based on different thermosensitive polymers used for culturing and detachment of skin cell. Two types of substrates with promising properties and used with good results as a scaffold for the culture of skin cells. Goal: The aim of study is to carry out the implementation of thermosensitive polymer coated scaffolds. Another goal of this project is to further modify and improve the properties of the matrix, as well as adapt them to the requirements of products ready to implement. Methods: The tested scaffolds are based on polymers: oligoethylene glycol methacrylate (PTEGMA) and 2-substituted-2- oxazoline(POX). Optimization of the culture process, cell adhesion and detachment of the sheet will be achieved by using cells isolated from the patients' skin. Result: In order to standardize the culture methods and detachment of sheets of fibroblast culture studies were conducted on the thermosensitive polymer PTEGMA, coated on glass discs. Detachment of the cells in defined temperature was performed. As part of a study was selection of a device for transferring a sheet of cells; synthetic skin (Suprathel) and hydrogel dressing (Aqua-Gel) were examined. Study on survival of transferred cells was performed. Conclusion: It appears that the time of fibroblast culturing has no effect on the cells detachment from the tested scaffold. Cells detached as a sheet, after 24 hours of culture, as well as after 148 hours of culturing. Temperature 17°C is suitable for cell detachment procedure independently on the time of culture. The decisive factor in this case was the number of cells. In the research of transferring tools, both Suprathel and Aqua-Gel are a good tool to transfer the sheets of cells to a desired location such as a wound. The results of the study also showed that the cells survived the procedure of the transfer and retain the ability to adhere to the culturing bottle, proliferation and colony formation. Leaving the cells stuck to the tested dressings over the wound does not adversely affect their growth. However, in the case of Aqua-Gel, some of the cells are entrapped into gel. This work was supported by the National Centre for Research and Development, project POLYCELL PBS1/B9/10/2012.

214

P-052 – Celluralization of membranes for guided bone regeneration with osteoblasts derived from bone marrow mesenchymal stem cells – a pilot study E. Olender,1 F. Dąbrowski,2 J. Olkowska-Truchanowicz,1 I. Uhrynowska-Tyszkiewicz,1 A. Kaminski,1 A. Wojtowicz3

1 Medical University of Warsaw, Department of Transplantology and Central Tissue Bank; National Centre for Tissue and Cell Banking, Warsaw, Poland 2 Medical University of Warsaw, 1st Department of Obstetrics and Gynecology, Warsaw, Poland 3 Medical University of Warsaw, Department of Dental Surgery, Warsaw, Poland

Introduction: Guided bone regeneration (GBR) is mainly used to repair bone defects in maxilla and mandible. Application of membranes in GBR techniques aims at an effective separation of bone defect site from the soft tissue ingrowth. Creation of such a barrier improves de novo bone formation by osteoprogenitor cells migrating to the defect site. The function of GBR membranes can be compared with the function of periosteum as an external lining sheet. The fundamental difference is the lack of cells (including osteogenetic stem cells) in the membranes. Goal: The aims of the study were to examine: 1) the possibility of celluralization of GBR membranes with human osteoprogenitor cells; 2) the influence of the membrane on the osteogenetic phenotype of cells seeded. Methods: Human osteoblasts derived from bone marrow mesenchymal stem cells were seeded onto the following materials: resorbable collagen sheet, collagen sponge, pericardium, amniotic membrane, cancellous bone allograft – 4x105 for each sample. Seeded materials were incubated in 37°C, 5% CO2 for 6 days. Osteogenetic differentiation promoting culture medium was used. Phenotype of cells was checked by real-time PCR method – osteogenetic gene expression profile. As controls unseeded materials incubated under the same conditions were used. Results: Osteoblasts derived from bone marrow mesenchymal stem cells adhere, colonize and maintain viability as well as their phenotype on materials used in the study. Detailed results will be presented during the congress. Conclusion: GBR membranes may be effectively enriched with cells. Cellularization with osteoblasts might have an improving effect on bone formation, as it is in case of revitalization of bone allografts used for bone augmentation and already clinically applied.

215

P-053 – Culturing and storing different human skin cell types to repopulate decellularized human dermal matrices G. Verween,1 J.-P. Draye,1 M. A. L. M. Boone,2 A. Aiti,3 G. Verbeken,1 D. De Vos,1 T. Rose,1 S. Jennes,1 V. del Marmol,2 G. Jemec,4 J.-P. Pirnay1

1 Human Cell and Tissue Banks, Laboratory for Molecular and Cellular Technology, Burn Wound Centre, Queen Astrid Military Hospital, Brussels, Belgium 2 Department of Dermatology, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium 3 Emilia Romagna Regional Skin Bank and Cell Factory, Burn Center Bufalini Hospital, Cesena, Italy 4 Department of Dermatology, Roskilde Hospital, Health Sciences Faculty, University of Copenhagen,

Denmark

In our laboratory, neonatal foreskin keratinocytes are cultured in a defined and animal product-free cell culture environment. These allogeneic keratinocytes are used to stimulate wound healing either as sheets for temporarily coverage of donor sites or cell-sprayed on meshed skin autografts. For permanent burn wound coverage, patients are treated with autologous keratinocyte cultures. But in third degree burns, due to the lack of an appropriate support, the adhesion of the cultured epithelial autografts is not satisfactory. The purpose of the study is to cultivate different autologous cell types, keratinocytes, fibroblasts, melanocytes and adipose-derived stem cells (ADSC), in order to evaluate the achievability of in vitro repopulating decellularized human dermal matrices (DHDMs). The different cells types were isolated from human adult donors and cultured for several passages in appropriate culture media. For storage, cells were cryopreserved. The cryopreserved cells were used to repopulate the DHDMs. The DHDMs were prepared from cryopreserved allogenic human skin (0.2-0.4 mm thick) by using a two step procedure. In a first step the epidermis was removed by a hypertonic treatment (1M NaCl) and in a second step a detergent treatment (0.5% Triton X-100) was used to remove the cellular debris in the dermis. Cell repopulation of the DHDMs was evaluated by histology and MTT assay. The different cell types were successfully isolated and cultured for several passages. Cryopreserved cells were either sub-cultured in 6-well plates or seeded on DHDMs (50,000 cells/cm²). A sequential seeding approach was used, fibroblasts or ADSC were first seeded on the reticular dermis side of the DHDMs and a week later keratinocytes were seeded on the papillary side of the DHDMs. Preliminary results indicate that the DHDMs can be cell repopulated. For recellularisation of DHDMs by different cells types care must be taken in choosing the appropriate properties of DHDMs for the development of living skin equivalents.

216

P-054 – Multilineage differentiation potential of human amniotic membrane in toto S. Wolbank,1,2,3 A. Lindenmair,1,2 A. Banerjee,1,2 S. Nürnberger,1,2 C. Hackl,2,4 S. Hennerbichler-

Lugscheider,2,4 C. Gabriel,2,4 J. Eibl,5 H. Redl1,2

1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Linz/Vienna, Austria 2 Austrian Cluster for Tissue Regeneration, Austria 3 Trauma Care Consult, Vienna, Austria 4 Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria 5 Bio-Products & Bio-Engineering AG, Vienna, Austria

Human amniotic membrane (hAM), already applied in many clinical settings, is also becoming interesting for new areas including tissue engineering. Applying tissue engineering strategies, isolated cells are typically combined with suitable carrier materials. Alternatively, the so called cell sheet technology enables transplantation without the use of carrier substances. Concept of our study is to differentiate amniotic membrane, which constitutes a pre-formed sheet of stem cells, in toto without any cell isolation. Our aim was to study a new approach for bone, cartilage and nerve tissue engineering via differentiation of amnion sheet. Therefore, hAM biopsies were cultured under conditions including either osteogenic, chondrogenic or Schwann cell differentiation. The resulting hAM was characterized for lineage-specific parameter including mineralization and calcium content, (immuno)histochemical analysis, quantification of glycosaminoglycans (GAG), qRT PCR for specific mRNAs as well as ELISA for neurotrophic factors at different time points. All differentiation strategies resulted in upregulation of the analyzed lineage-specific markers. Under osteogenic conditions mineralization was observed and most of the cells expressed osteocalcin after 28 days. These results were confirmed by increased calcium-content and mRNA expression of specific osteogenic markers. GAG production was induced under chondrogenic conditions, especially within an induction medium containing FGF-2. These findings were further attended by quantification of the GAG content, and by upregulation of chondrogenic markers at mRNA level. When inducing a Schwann cell-like phenotype in hAM, levels of neurotrophic factors were increased, while the cells took up a glia-like morphology. Thus, these in-vitro results are promising steps towards a versatile use of living hAM as a natural biomaterial for tissue engineering.

217

P-055 – National information technology in eye banking - from donor to recipient M. Nasic

University Hospital Center Zagreb, Zagreb, Croatia

Introduction: Croatia implemented health information technology in national cornea tissue transplant program. The IT system was created in conditions of assisting the Ministry of Health, well established professional societies and the lack of institutions for quality assurance in health care. Goal: To present the results of two years of monitoring the implementation of national IT program that covers the cornea tissue from the donor to the recipient, unexpected challenges and benefits of administration.

Methods: We analyzed the time and number of tissue required for the quality establishment of IT program and the quality and the quantity of completed forms in the first and second year of implementation by teams. Identified was benefits obtained by using the IT base, compared to the paper records and suggest possible solutions to identified barriers. Results: Creating a professional concept of IT database and communication between teams lasted 5 months. Partner to Ministry of Health was Committee for Promotion of Transplantation Program of the Croatian Ophthalmological Society. Professional IT company created computer program 4 months. Pilot project of IT database lasted a month. The duration of the pilot project was too short and the weaknesses the IT database application was noticed another 7 months after completion of the pilot project. The reason may be the small number of retrieval corneas, an average of 15 per month, so that all the weaknesses of the IT database may notice after 100 retrieval and 50 transplanted corneas. The IT program includes 12 IT forms with 1160 data including national waiting list. The quality indicators can be monitoring daily. The records are filled (first year- second year) with following percentage: procurement coordinators – 92% -98%, retrieval teams – 76%-82%, eye bank 98%-100% i transplant teams 83%-93%. Conclusion: Although ensuring a factor was binding decision of the Minister of Health that the IT database is only official database, there is a high level of satisfaction with IT base by users (100%).

218

P-056 – Comparison of cell viability and expression of stem cell markers in fresh and stored limbal tissue F. Keshtkar,1 S. Mason,1 P. Rooney,2 E. Austin,2 R. Stewart, S. Kaye,1 C. Sheridan1 1 Department of Eye and Vision Sciences, University of Liverpool, Liverpool, UK 2 NHS Blood and Transplant, Liverpool, UK.

The limbus is crucial in regenerating the cornea and maintaining vision. Current guidelines for limbal transplant dictate that donor limbal tissue must be obtained within 72 hours of death if an allograft from a living donor is not available. Previous research has suggested better outcomes for whole limbal allografts obtained from fresher material. The present aim was to assess the effect of eye bank storage on limbal cells by analysing total live and dead cell counts and using markers for stem cells to compare levels in fresh and stored samples. Fresh samples were found to have a significantly greater percentage of live cells compared with stored cells (mean 39.8 ±8.3 vs. 8.7 ±3.0, p <0.001). Immunocytochemical staining using TrkA showed a significantly lower proportion of positive cells in the fresh sample (68.8% ±2.2 vs. 83.1% ±1.4, p<0.01), while staining with p63 showed converse results (32.9% ±6.0 vs. 17.3% ±7.8, p>0.05). A TUNEL assay demonstrated a slightly higher proportion of apoptotic cells in stored tissues (3.4% ±1.4 vs. 1.7% ±0.7, p >0.05). In conclusion, stored cells may retain the ability to maintain stem cells, and therefore have the potential to produce an epithelial layer. Further work is required in determining the stem cell population and the effects of donor age, death to organ retrieval time and time in organ culture.

219

P-057 – Group project of international on-line course in tissue banking, cell therapy & regenerative medicine: raising awareness in bone donation C. Hynes,1 L. Flores,2 M. J. Hidalgo Rus,3 M. Pizi,4 V. Tobar Roa,5 M. Istrate,3 S. Mazić6 1 IBTS, Dublin, Ireland 2 Universidad de Barcelona, Barcelona, Spain

3 TPM – DTI Foundation, Hospital Clinic de Barcelona, Barcelona, Spain

4 Nicosia General Hospital, Nicosia, Cyprus

5 FOSCAL, Bucaramanga, Colombia

6 UHC Zagreb, Zagreb, Croatia

Introduction: The International On-line Course in Tissue Banking, Cell Therapy & Regenerative medicine offers education in tissue banking and cell therapy in response to a growing demand of tissue bank professionals. Students gathered from different settings spent two months learning in compliance with the professional requirements, current practices and recognized standards of proficiency in the field. Presented with a task of developing a group project this is what we have come up with. Goal: The aim of our project is to sensitize and inform the general public and health community about the importance of bone tissue donation. We want to emphasize how tissue donation can be life enhancing and change it s perception as being brutal or degrading to the body. Results: A planning process divided into ten stages will be applied. A leader, expert in the issue will be the manager of the project. A meeting with all the interested parties will be convened and an educational information campaign started. The project leader will identify specialist doctors and centres with an interest in participation. A gap analysis will be performed including human resources, the needed facilities, microbial and virology testing, financial costs, licensed regulation, ethical aspects, quality/validation requirements and training needs. A timeline of the working plan will be scheduled and followed. Staff training will be required and volunteer patients included. The campaign will include videos, pictures, real-life stories, existing donation channels and posters. Target audiences are, family members of recipient and post-mortem donors, living hip replacement donors and health care professionals. Annually the re-education of the staff and introduction of new staff will be organised. Conclusion: We hope to inform the general and medical population not just about organ donation, but about organs and tissues. This goal will be measured by an increase in consent and retrieval rates as well as an increase in the volume of tissue issued by the tissue bank.

220

Author index Abdalla E, 169 (P-009) Agusti E, 145, 181, 198 (O-063/P-019/P-036) Aiti A, 128, 215 (O-047/P-053) Akanyi N, 165, 166 (P-005/P-006) Alemani X, 181 (P-019) Alsina M, 173 (P-013) Amiot S, 150 (O-068) Anastasesku M, 188 (P-026) Anestakis D, 211 (P-049) André W, 147 (O-065) Aouassar N, 147 (O-065) Ashford P, 96 (O-018) Austin E, 218 (P-056) Bader B, 146 (O-064) Balleste C, 173 (P-013) Banerjee A, 216 (P-054) Barba M, 110, 118 (O-032/O-037) Bartlett S, 38, 79 (O-001) Barrera-L A, 175, 209, 210 (P-015/P-047/P-048) Barth J, 155 (O-072) Beguin Y, 113 (O-034) Beele H, 32, 100 (O-022) Benedek F, 96 (O-018) Berckmans D, 162 (P-002) Bergers S, 140, 171 (O-058/P-011) Barrera-L A, 189 (P-027) Bertazzo S, 84 (O-006) Bermudez Gonzalez T, 191 (P-029) Bialecki J, 141 (O-059) Bianchini A, 150 (O-068) Blancke F, 164 (P-004) Blanco F, 203, 204 (P-041/P-042) Bojanic I, 129 (O-048) Bokhorst A, 89, 108, 111, 140, 171 (O-011/O-030/O-033/O-058/P-011) Boogaerts M, 123 (O-042) Boone A, 128, 215 (O-047/P-053) Bortlik K, 185 (P-023) Braun C, 174 (P-014) Brennan M, 127 (O-046) Brockbank K, 57, 111 (O-033) Brubaker S, 45, 91 (O-013) Bellare J, 192 (P-030) Berkert J, 157 (O-074) Bujan J, 203, 204 (P-041/P-042) Campbell L, 111 (O-033) Carballal Rodriguez M, 191 (P-029) Casaroli R, 173 (P-013) Celis P, 63, 130 (O-049) Ceulemans C, 176, 178 (P-016/P-017) Chabannon C, 41, 86 (O-008) Chambon J-P, 150 (O-068) Chen Z, 111 (O-033) Chenorhokian H, 73, 150 (O-068) Cicione C, 110, 118 (O-032/O-037)

221

Ciobanu P, 168 (P-008) Cornu O, 147 (O-065) Crkvenac-Gornik K, 129 (O-048) Cuppens S, 147, 195 (O-065/P-033) Dabrowski F, 214 (P-052) Daniels S, 115 (O-035) Danze P-M, 144 (O-062) Davidovic S, 129 (O-048) Davies J, 83, 84 (O-005/O-006) D’Amato Tothova J, 135, 183, 184, 194 (O-054/P-021/P-022/P-032) De By T, 89 (O-011) De Corte P, 105 (O-027) De Gelas S, 164 (P-004) Dejaegher J, 162 (P-002) Del Marmol V, 128, 215 (O-047/P-053) De Toro J, 203, 204 (P-041/P-042) Deussen A, 185 (P-023) De Vleeshouwer S, 125, 162 (O-044/P-002) De Vos D, 90, 105, 128, 176, 178, 215 (O-012/O-027/O-047/P-016/P-017/P-053) Devos T, 123 (O-042) Diaz Prado S, 203, 204 (P-041/P-042) Diaz Rodriguez R, 136, 164 (O-055/P-004) Dierickx D, 162 (P-002) Di Stasio E, 118 (O-037) Di Taranto G, 110, 118 (O-032/O-037) D’Lima C, 192 (P-030) Domanovic D, 65, 132 (O-051) Domenech Garcia N, 205, 206, 207, 208 (P-043/P-044/P-045/P-046) Doser M, 52, 101 (O-023) Draye J-P, 90, 105, 128, 156, 176, 178, 215 (O-012/O-027/O-047/O-073/P-016/P-017/P-053) Dubois J, 115 (O-035) Dufrane D, 62, 118, 122, 126, 134, 147, 182, 195 (O-038/O-041/O-045/O-053/O-065/P-020/P-033) Durakovic N, 129 (O-048) Dürselen L, 193 (P-031) Ectors N, 31, 125, 176, 178 (O-044/P-016/P-017) Eibl J, 216 (P-054) El Khoury G, 74, 151 (O-069) Escobedo-G B, 175 (P-015) Eyrich M, 40, 85 (O-007) Fan Y, 136, 164 (O-055/P-004) Farinas O, 181 (P-019) Fehily D, 49, 95, 137 (O-018/O-056) Fellah M, 169 (P-009) Fernandez Mallo O, 191 (P-029) Ferreira P, 195 (P-033) Flores L, 219 (P-057) Fölsch C, 56, 107 (O-029) Fraga Marino M, 205, 206, 207, 208 (P-043/P-044/P-045/P-046) Fuentes-Boquete I, 203, 204 (P-041/P-042) Gabriel C, 202, 216 (P-040/P-054) Gafud N, 169 (P-009) Galli C, 122 (O-041) Garcia-C R, 189,190 (P-027/P-028) Garcia Garcia M, 191 (P-029) Gatto C, 135, 182, 184, 194 (O-054/P-021/P-022/P-032) Geesink I, 89 (O-011) Ghirardini A, 96 (O-018)

222

Gindraux F, 199 (P-037) Giurgola L, 135, 183, 184, 194 (O-054/P-021/P-022/P-032) Glik J, 121, 186, 200, 201 (O-040/P-024/P-038/P-039) Goebbels M, 122 (O-041) Godts B, 119 (O-038) Golemovic M, 129 (O-048) Golubic Cedulic B, 129 (O-048) Gorlitzer M, 142 (O-060) Grabenwöger M, 142 (O-060) Graw M, 174 (P-014) Grazka E, 109, 187 (O-031/P-025) Greene D, 111 (O-033) Grindraux F, 127 (O-046) Guerreschi P, 144 (O-062) Gut G, 109, 187 (O-031/P-025) Gutierrez J, 188 (P-026) Guzman-M K, 175, 189, 209 (P-015/P-027/P-047) Habramova A, 157 (O-074) Hacki C, 216 (P-054) Happel M, 140, 171 (O-058/P-011) Harder M, 54, 103 (O-025) Hatzfeld A-S, 144 (O-O62) Haun M, 180 (P-018) Heemskerk M, 82 (O-004) Heerings M, 89 (O-011) Hendrikx M, 115 (O-035) Hennerbichler-Lugscheider S, 202, 216 (P-040/P-054) Hensen K, 115 (O-035) Hermida-Gomez T, 203, 204 (P-041/P-042) Herrlinger F, 163 (P-003) Hetsch N, 148 (O-066) Hidalgo Rus J, 219 (P-057) Hildebrandt M, 51, 98 (O-020) Hoff-Lenczewska D, 121, 186, 200, 201 (O-040/P-024/P-038/P-039) Hohenberger P, 120 (O-039) Hownietz N, 142 (O-060) Huam M, 81 (O-003) Huma I, 129 (O-048) Huys I, 176, 178 (P-016/P-017) Hynes C, 219 (P-057) Ignatius A, 193 (P-031) Ilic I, 129 (O-048) Isgro A, 110, 118 (O-032/O-037) Istrate M, 172, 173, 219 (P-012/P-013/P-057) Jamaer L, 115 (O-035) Jashari R, 30, 104, 136, 149, 164, 165, 166 (O-025/O-055/O-067/P-004/P-005/P-006) Jares L, 153 (O-071) Jastrzebska A, 109, 187 (O-031/P-025) Jennes S, 90, 105, 128, 156, 176, 178, 215 (O-012/O-027/O-047/O-073/P-016/P-017/P-053) Jimenez-O F, 209 (P-047) Jemec G, 128, 215 (O-047/P-053) Johnson M, 83, 84 (O-005/O-006) Jung S, 193 (P-031) Kafu E, 169 (P-009) Kalab M, 170, 197 (P-010/P-035) Kaltenbrunner W, 146, 155 (O-064/O-072) Kaminski A, 109, 161, 187, 214 (O-031/P-001/P-025/P-052)

223

Karkoska J, 170, 197 (P-010/P-035) Kaudela K, 108, 146, 155 (O-030/O-064/O-072) Kawecki M, 121, 186, 200, 201 (O-040/P-024/P-038/P-039) Kaye S, 218 (P-056) Keshtkar F, 218 (P-056) Klama-Baryla A, 121, 186, 200, 201, 213 (O-040/P-024/P-038/P-039/P-051) Klatte-Schulz F, 212 (P-050) Klykens J, 99, 123 (O-021/O-042) Knoop S, 185 (P-023) Kobylka P, 157 (O-074) Koninckx R, 115 (O-035) Koppen C, 116 (O-036) Kraut M, 121, 186, 200, 201 (O-040/P-024/P-038/P-039) Kriner F, 174 (P-014) Kumar A, 192 (P-030) Labar B, 129 (O-048) Labus W, 121, 186, 200, 201 (O-040/P-024/P-038/P-039) Laenen A, 123 (O-042) Lannau B, 100 (O-022) Lattanzi W, 110, 118 (O-032/O-037) Laurent R, 127, 199 (O-046/P-037) Layrolle P, 127, 199 (O-046/P-037) Leichtle A, 53, 102 (O-024) Lejuste P, 119 (O-038) Lekeux L, 134, 182 (O-053/P-020) Lerut J, 71, 148 (O-066) Leysen L, 116 (O-036) Lindenmair A, 202, 216 (P-040/P-054) Link D, 108 (O-030) Limongelli A, 135 (O-054) Lobo Gajiwala A, 192 (P-030) Lomas R, 131 (O-050) Lomelli-R C, 175 (P-015) Lonsky V, 170, 197 (P-010/P-035) López Fraga M, 50, 97 (O-019) Lopez Pinon M, 191 (P-029) Lotherington K, 81, 180 (O-003/P-018) Lugmayr E, 202 (P-040) Luque S, 181 (P-019) Mabrouk A, 169 (P-009) MacDonald P, 131 (O-050) Macianskiene R, 115 (O-035) Machuca-R C, 189, 209, 210 (P-027/P-047/P-048) Madinaveitia-V A, 175, 189, 190, 209, 210 (P-015/P-027/P-028/P-047/P-048) Maj M, 121, 200 (O-040/P-038) Manyalich M, 172, 173 (P-012/P-013) Marais A, 195 (P-033) Mareri M, 96 (O-018) Marowska J, 109, 187 (O-031/P-025) Marczińsky W, 68, 141 (O-059) Marquetti P, 144 (O-062) Marsit N, 169 (P-009) Martinache I, 55, 106, 144 (O-028/O-062) Martinez-F F, 175, 189, 190, 209, 201 (P-015/P-027/P-028/P-047/P-048) Martinez N. Otero E, 145 (O-063) Martinez-Conesa M, 198 (P-036) Martinez-Sanchez A, 203, 204 (P-041/P-042)

224

Mason S, 218 (P-056) Massouille D, 150 (O-068) Matejkova A, 170 (P-010) Mazic S, 129, 219 (O-048/P-057) Mees U, 115 (O-035) Meinhart J, 142 (O-060) Merni X, 134, 182 (O-053/P-020) Meyns B, 76, 158 (O-075) Michael Real J, 153 (O-071) Michetti F, 110, 118 (O-032/O-037) Miguel Torres I, 191 (P-029) Mikulic M, 129 (O-048) Misto R, 135 (O-054) Mohr J, 81, 180 (O-003/P-018) Mubagwa K, 115 (O-035) Mujaj B, 136 (O-055) Myint P, 167 (P-007) Nacu V, 168 (P-008) Nallet A, 199 (P-037) Nani Costa A, 96 (O-018) Nasic M, 217 (P-055) Ngakam R, 136, 164, 165, 166 (O-055/P-004/P-005/P-006) Nicod L, 127, 199 (O-046/P-037) Noël L, 39, 80, 93 (O-002/O-015) Nowak M, 121, 186, 200, 201 (O-040/P-024/P-038/P-039) Nürnberger S, 216 (P-054) Obert L, 127, 199 (O-046/P-037) Olender E, 161, 214 (P-001/P-052) Oliva R, 181 (P-019) Olkowska-Truchanovicz J, 214 (P-052) Otero N, 198 (P-036) Parsons C, 81 (O-003) Pascual B, 36, 105 (O-027) Pasquesoone L, 144 (O-062) Pateri F, 135 (O-054) Pauwels F, 162 (P-002) Petanidis S, 211 (P-049) Perez M, 145, 181, 198 (O-063/P-019/P-036) Perot C, 150 (O-068) Phillips G, 167 (P-007) Pinda Y, 167 (P-007) Pinto de Carvalho D, 195 (P-033) Pipeleers D, 60, 124 (O-043) Pirnay J-P, 33, 90, 105, 128, 156, 176, 178, 215 (O-012/O-027/O-047/O-073/P-016/P-017/P-053) Pitt T, 64, 131 (O-050) Pittie M, 43, 88 (O-010) Pizi M, 219 (P-057) Pless-Petig G, 185 (P-023) Pocobelli A, 135 (O-054) Pomar J, 173 (P-013) Ponte Valesco N, 191 (P-029) Ponzin D, 70, 143 (O-061) Possemiers T, 116 (O-036) Prat A, 75, 152 (O-070) Pruss A, 163, 193, 212 (P-003/P-031/P-050) Qassemyar P, 144 (O-062) Raftopulos C, 119 (O-038)

225

Reichel H, 193 (P-031) Rauen U, 185 (P-023) Razborschii V, 168, (P-008) Rose T, 69, 90, 105, 128, 156, 176, 178, 215 (O-012/O-027/O-047/O-073/P-016/P-017/P-053) Rossel L, 174 (P-014) Rössner E, 120 (O-039) Redl H, 216 (P-054) Rendal-Vazquez M, 191, 203, 204, 205, 206, 207, 208 (P-029/P-041/P-042/P-043/P-044/P-045/P-046) Rooney P, 218 (P-056) Rummens J-L, 58, 115 (O-035) Saegeman V, 66, 133 (O-052) Salagarello M, 110, 118 (O-032/O-037) Salifoglou A, 211 (P-049) Samant U, 192 (P-030) Sanchez Ibanez J, 205, 206, 207, 208 (P-043/P-044/P-045/P-046) Sandoval-Z H, 189, 190, 210 (P-027/P-028/P-048) Sanjurjo-Rodriguez C, 203, 204 (P-041/P-042) Sanlius Verdes N, 205, 206, 207, 208 (P-043/P-044/P-045/P-046) Sanlius Verdes A, 206, 207, 208 (P-044/P-045/P-046) Sarikouch S, 77, 159 (O-076) Savio A, 181 (P-019) Scally E, 194 (P-032) Scheffler S, 212 (P-050) Schenner C, 146 (O-064) Schmidt T, 212 (P-050) Schoemans H, 123 (O-042) Schroeter J, 163 (P-003) Schubert T, 119, 122, 134 (O-038/O-041/O-053) Schuchard Y, 196 (P-034) Selvais S, 119 (O-038) Sefert M, 111 (O-033) Segur J, 173 (P-013) Seija V, 188 (P-026) Semiglia G, 188 (P-026) Serventi-Seiwerth R, 129 (O-048) Shaw S, 194 (P-032) Sheridan C, 218 (P-056) Sinap F, 123 (O-042) Sing A, 192 (P-030) Sinha N, 192 (P-030) Siska I, 48, 95 (O-017) Skiific M, 129 (O-048) Smith M, 120, 162 (O-039/P-002) Solie L, 162 (P-002) Spatenka J, 157 (O-074) Steels P, 115 (O-035) Steijger P, 196 (P-034) Stein J, 89 (O-011) Stewart R, 218 (P-056) Stock A, 111 (O-033) Stumpflen A, 142 (O-060) Tassignon M-J, 59, 116 (O-036) Temple L, 153 (O-071) Teotia R, 192 (P-030) Teskrat F, 67, 138 (O-057) Theiss D, 202 (P-040) Tobar R, 219 (P-057)

226

Trias E, 46, 92, 145, 181, 198 (O-014/O-063/P-019/P-036) Tsolaki M, 211 (P-049) Uhrinowska-Tyszkiewicz I, 72, 109, 149, 161, 187, 214 (O-031/O-067/P-001/P-025/P-052) Urbanowska E, 161 (P-001) Ustoa S, 175 (P-015) Valster H, 108 (O-030) Van Brummelen S, 82 (O-004) Vandenschrik V, 134, 182 (O-053/P-020) Van der Spiegel S, 42, 87 (O-009) Vanderkelen A, 90, 105, 176, 178 (O-012/O-027/P-016/P-017) Vandeynse D, 182 (P-020) Van Geyt C, 37, 100 (O-022) Van Gool S, 61, 125, 162 (O-044/P-002) Van Hoeck B, 136, 164, 165, 166 (O-055/P-004/P-005/P-006) Van Leiden H, 82 (O-004) Van Maele G, 100 (O-022) Van Marion W, 196 (P-034) Van Steenberghe M, 122, 134 (O-041/O-053) Vartic D, 168 (P-008) Verbeken G, 44, 90, 105, 128, 156, 176, 178, 215 (O-012/O-027/O-047/O-073/P-016/P-017/P-053) Verhelpen M, 119 (O-038) Verween G, 105, 128, 215 (O-027/O-047/P-053) Vespasiano M, 96 (O-018) Vilaronda A, 145, 181, 198 (O-063/P-019/P-036) Vitacolonna M, 120 (O-039) Vito S, 181 (P-019) Visconti G, 118 (O-037) Vizcaino P, 175 (P-015) Vobornik M, 157 (O-074) Vojacek J, 157 (O-074) Warwick R, 47, 94 (O-016) Wells C, 153 (O-071) Wesinger M, 146 (O-064) Wiersum-Osselton J, 140 (O-058) Wildemann B, 212 (P-050) Wilkemeyer I, 163 (P-003) Windmolders S, 115 (O-035) Witzeneder K, 202 (P-040) Wojtowicz A, 214, 161 (P-001/P-052) Wolbank S, 216 (P-054) Wondergem J, 167 (P-007) Yebra-Pimental Vilar M, 205, 206, 207, 208 (P-043/P-044/P-045/P-046) Zacek P, 157 (O-074) Zakaria N, 116 (O-036) Zatschler B, 185 (P-023) Zijlker-Jansen J, 140, 171 (O-058/P-011) Zunino J, 188 (P-026)

227

228

Organisation

Organising Committee

Congress President

Ramadan Jashari, MD, FETCS

Members

Hilde Beele, MD, PhD Pierre Neirinckx, Col, MD, SBH Chris Deroubaix, Lt Col, MAB Bruno Pascual, MSc Eng

Bruno Deschans, MSc Jean-Paul Pirnay, MSc Eng, PhD Nadine Ectors, MD, PhD Caroline Van Geyt, MSc Ye Dong Fan, MD, PhD Gilbert Verbeken, MSc

Johan Guns, MSc Harry Vindevogel, Maj Gen, MSc Eng Ludo Muylle, MD, PhD

Scientific Committee

Chair

Jean-Paul Pirnay, MSc Eng, PhD

Members

Etienne Baudoux, Belgium John Kearney, UK Hilde Beele, Belgium Ján Koller, Slovakia

Arlinke Bokhorst, The Netherlands Johan Kurz, Austria Martin Börgel, Germany Peter Lodewyckx, Belgium

Scott Brubaker, USA Torsten Malm, Sweden Carl Ceulemans, Belgium Dimitri Mikhalski, Belgium

Jill Davies, UK Hans-Joachim Mönig, Germany Theo De By, The Netherlands Aurora Navarro Martinez-Cantullera, Spain

Daniel De Vos, Belgium Richard Ngakam Noumanje, Belgium Timothy Devos, Belgium Robert Parker, UK

Jean-Pierre Draye, Belgium Axel Pruss, Germany Denis Dufrane, Belgium Thomas Rose, Belgium

Ted Eastlund, USA Johan Somville, Belgium Nadine Ectors, Belgium Jaroslav Spatenka, Czech Republic Patrick Evrard, Belgium Esteve Trias, Spain Ye Dong Fan, Belgium Izabela Uhrynowska-Tyszkiewicz, Poland Deirdre Fehily, Italy Ivan Van Riet, Belgium

Simone Hennerbichler-Lugscheider, Austria Alain Vanderkelen, Belgium Isabelle Huys, Belgium Gilbert Verbeken, Belgium

Ramadan Jashari, Belgium Ruth Warwick-Cohen, UK Artur Kaminski, Poland