bioreactors (1)
DESCRIPTION
Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011. - PowerPoint PPT PresentationTRANSCRIPT
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
BIOREACTORS (1)
Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 5
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Static cell cultures• Most frequently applied cell culture method• Petri dishes or tissue culture flasks• Adherent cells: monolayer cultures• Suspension cells: relatively lower densities
Advantages: no special equipment required, relatively cheap and easyDrawbacks: lower cell density, lower metabolism rate
TÁMOP-4.1.2-08/1/A-2009-0011
Problems concerning static cell cultures• Lack of vascularization• Nutrient supply is limited• Oxygen supply is limited• Metabolic end-product removal is problematic• Frequent and regular passage required• Periodic medium change needed• In vivo dynamic tissue and cellular
environment is physiological
TÁMOP-4.1.2-08/1/A-2009-0011
Bioreactors: dynamic cell environment• Dynamic and continuous nutrient and oxygen
supply• Possibility of formation 3D tissue structure• Increasing cell-cell contact possibilities• Mechanical stimulation of cell cultures• May promote cellular differentiation in the
desired direction• Markedly higher cell densities can be
achieved• Higher cell density allows large scale
industrial application of cell cultures
TÁMOP-4.1.2-08/1/A-2009-0011
Mass transport challenges in 3D tissue culturesDiffusion of oxigen and nutrients:• From the static medium to the surface cells• From the surface cells to the deeper
structuresImortant parameters of the cultured cell/tissue
construct:• Porosity• TortuosityTissue thickness under static conditions should not exceed 100 mm
TÁMOP-4.1.2-08/1/A-2009-0011
Shear forces in dynamic fluidsShear stress measure unit:dyn/cm2
1 dyn = 10mN
A shear stress, t is applied to the top of the square while the bottom is held in place.
t
l
Dx
TÁMOP-4.1.2-08/1/A-2009-0011
Shear stress in bioreactors• Shear stress distribution is uneven in
bioreactors• Highest stress is located around edges and
sides of the moving vessel• Design of bioreactors must aim evenly low
shear stress in the vessel• Uneven shear stress distribution affect cell
survival, density, proliferation, etc • Maximum shear stress for mammalian cells
are 2.8 dyn/cm2
TÁMOP-4.1.2-08/1/A-2009-0011
Cell distribution in dynamic environment• Uneven cell distribution in 3D constructs• Gradually decreasing cell density towards the
central area• Cell seeding problems• Diffusion problems• Challenges creating viable 3D tissues
TÁMOP-4.1.2-08/1/A-2009-0011
Bioreactor design requirements IThe aim of using bioreactors for TE is to overcome the hinders of static culture conditions.Bioreactors need to fulfill at least one of the following requirements:
1. Need to maintain desired nutrient and gas concentration in 3D constructs
2. Need to facilitate mass transport into 3D cultures3. Need to improve even cellular distribution in 3D
constructs4. Need to expose the construct to physical stimuli5. Need to provide information about the formation of
3D tissue
TÁMOP-4.1.2-08/1/A-2009-0011
Bioreactor design requirements II• Design should be as clear and simple as possible• Avoid structural recesses (risk of infection, cleaning
difficulty)• Simple and quick assembly and disassembly• Use of biocompatible or bioinert materials (no
chromium alloys or stainless steel)• Withstand heat or alcohol sterilization and humid
atmosphere• Proper embedding of instruments (e.g. thermometer,
pH meter, pump, rotator motor, etc.)
TÁMOP-4.1.2-08/1/A-2009-0011
Structure of an industrial bioreactor
Peristaltic pumps
Drive
Water in
Water out
Air
Counterpressure
valve
Electromagnetic
valve for cooling
Pump
Safetyvalve
Process controller
Heater vessel
Acid BaseAntifoam Substrate
Q
Q valve
FoamT
pHpO2
TÁMOP-4.1.2-08/1/A-2009-0011
Spinner flask bioreactors• Stirred fluid, suspended
cells, fixed scaffolds • Eddy around the edges of
scaffolds• These small, turbulent
flows enhance cell seeding and mass transport into the scaffolds
• Typically, stirring speed is 60-80 rpm, volume 120-8000 ml, 50% medium change in every two days
• TE cartilage grown to 0.5mm thick under these conditions
Spinner flask bioreactor
TÁMOP-4.1.2-08/1/A-2009-0011
Rotating wall bioreactors I
Fc Fd
Fg
TÁMOP-4.1.2-08/1/A-2009-0011
Rotating wall bioreactors II• Originally developed by NASA to study cell cultures
at high g accelerations during space flight • Widespread application in Earth surface• Scaffolds are free to move in the media• Constant angular speed of rotation is ensured• The hydrodynamic drag force balances the gravity
ensuring the constant suspension of the scaffold in the medium
• Medium change may be either constant or intermittent
• RWV provides similar fluid transport and homogenous cell distribution like those in the spinning flask
TÁMOP-4.1.2-08/1/A-2009-0011
Compression bioreactors I
Head for dispensing pressure
Scaffold constructs
TÁMOP-4.1.2-08/1/A-2009-0011
Compression bioreactors II • Main use for cartilage TE• Static or dynamic pressure can be applied• Motor generating linear motion force• Linear displacement sensors • Load is transferred to the cell seeded constructs via
flat platens• Even load distribution is critical• A special method of transferring pressure to cartilage
constructs is to use hydrostatic pressure bioreactors
BIOREACTORS (2)
Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 6
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Strain bioreactors• Tendon, ligament, bone, cartilage and
cardiovascular tissue• Constant or pulsating power transfer• Tissue constructs are anchored to the power
transfer apparatus on an elastic basis • Strain is applied to the elastic basis and
transferred to the tissue construct
TÁMOP-4.1.2-08/1/A-2009-0011
Flow perfusion bioreactors I
Scaffold constructs with seeded cells
TÁMOP-4.1.2-08/1/A-2009-0011
Flow perfusion bioreactors II• Mass transport and nutrient delivery to cells
is similar to that of in vivo • Because of perfusion pressure, not only
diffusion but also convection contributes to oxigen delivery
• Mass transport distance is increased in flow perfusion bioreactors
• Medium perfusion can be used for cell seeding too
TÁMOP-4.1.2-08/1/A-2009-0011
Cartilage: tissue features and injury repair• Cartilage is an ECM-rich tissue, chondrocytes
secreting chondroitin-sulphate, collagen, elastic fibers, etc.
• Avascular tissue, nutrition is available through diffusion only
• Chondrocytes exert low metabolic activity and severe damage can not be restored in avascular tissue.
• Cartilage repair results in fibrous cartilage of poor mechanical properties
• In vivo body weight and joint motion exerts dynamic load on hyaline cartilage covering joint surfaces
TÁMOP-4.1.2-08/1/A-2009-0011
Compression bioreactors for cartillage TE I• Cartilage tissue aggregate modulus is no more
than 40% of native tissue in static cultures • Dynamic load can increase modulus of TE
cartilage near to the physiological value• Dynamic load increases ECM production of
chondrocytes• Addition of growth factors (TGF-b) also helps
chondrocyte differentiation• Compression loading is much more effective
promoting chondrocyte differentiation than TGF-b
TÁMOP-4.1.2-08/1/A-2009-0011
Compression bioreactors for cartillage TE II• For bioengineering functional load-bearing
tissues, like cartilage or bone, mechanical load has to be applied in the bioreactor.
• These forces are needed to express mechanosensitive Ca2+ channels, rearrangement of the cytoskeleton, and also MSC need mechanical strain to direct the differentiation
• Problems: mechanical parts are prone for leakage, infection
• Scaffolds have to withstand mechanical stimulation, so strong scaffolds are needed, which may have longer degradation time, which is not preferred
TÁMOP-4.1.2-08/1/A-2009-0011
Tissue Engineering in bone repair• Bone defect and non-union healing• Speeding up the process• Autologous or allogenic bone grafts• Xenograft trialsThese methods are associated with donor site morbidity, chronic pain, disease transmission, infetions
TÁMOP-4.1.2-08/1/A-2009-0011
Flow perfusion bioreactors in bone engineering• Flow perfusion bioreactors proved to be
superior compared to rotating wall or spinner flask bioreactors
• ALP, Osteocalcin and Runx2 expression is higher
• Scaffold mineralization is higher• Careful setting of flow rate because of
disadvantageous effects of high shear stress• Intermittent dynamic flow is more favourable
than steady speed flow
TÁMOP-4.1.2-08/1/A-2009-0011
Two chamber bioreactor• Two separate chambers• Simultaneous culture of different cell types• Application: generation of human trachea
TÁMOP-4.1.2-08/1/A-2009-0011
Current achievements in bioreactor designTE human trachea for implantation: • Decellularized donor trachea were seeded
with autologous chondrocytes and airway epithelium
• Two separate „chambers” allowed simultaneous culture of different cells
• Surgical replacement of the narrowed trachea in a TB patient
TÁMOP-4.1.2-08/1/A-2009-0011
Limitations of current bioreactors• Labor intensive methods• Current bioreactors are specialized devices• Difficult assembly and disassembly• Low cell output • Real-time monitoring of tissue structure and
organization is not available