CryopreservationSlow Freezing

Patrick Quinn PhD, HCLDpatrick.quinn@coopersurgical.com

Sage In-vitro Fertilization, Inc

Redmond, Oregon, USA

DISCLOSURE

I am Vice President of research and Development at Sage In-Vitro Fertilization, Inc.

We produce a range of commercial ART media products

Sage IVF

Production @

Pasadena

Redmond

Cooper Surgical,Trumbull

Sage IVF

Southern California

Central Oregon

Slow-freezing method

Vitrification

Attention to the size

Ice Crystal Ice Crystal FormationFormation

EquilibratiEquilibrationon

SeedingSeeding Slow Slow freezing freezing

Slow cooling requires a minimum of ~140 minutes.

Greatest problems:

extra & intracellular ice causing fracture osmotic swelling & shrinkingsolution effectchemical toxicity of CPAsexpensive equipment

Cryoprotectants

• The mechanism of the protective action of cryoprotectants is the same, but their toxicities are different;

• Permeability is different with different cryoprotectants and temperatures;

• Therefore, the toxicity of cryoprotectants must be considered for freezing;

• There are osmotic change before and after freezing in cryopreservation solution;

• These osmotic changes may cause the death of cells, normally it is referred to ‘osmotic injury’;

Cryoprotectants

Thawing Solutions

• Hypertonic solution is required, i.e. non-permeable sucrose is added to prevent swelling and shrinkage of the cells;

• Chilling injury;

• Cryoprotectant (toxicity and temperature);

• Osmotic injury;

• Speed of freezing and thawing;

Factors affecting successful frozen-thawing

Step by Step Use of Sage Embryo Cryopreservation

Media

Cryopreservation of 2PN/Cleaving Embryos/Blastocysts

QA Embryo Freeze Kit (ART-8014)

PPD, PPD+0.1M sucrose, Diluent – all with 12 mg/mL HSA.

QA Blastocyst Freeze Kit (ART-8015)

5% Glycerol, 9% Glycerol + 0.2 M Sucrose, Diluent

QA Thaw Kit (ART-8016)

0.5 M Sucrose, 0.2 M Sucrose, Diluent

Freezing of Zygotes, D2 & D3 Embryos

Use QA Embryo Freeze Kit ART-8014.

Prepare 0.5 M & 1.0 M propanediol (PPD) solutions.

Move embryos through solutions in following sequence.

0.9 mL/well + oil, 37oC.

PPD Concentration mL 1.5 M PPD mL Diluent

0.5 M 0.3 mL 0.6 mL

1.0 M 0.6 mL 0.3 mL

1 2

3 4

1. 0.5 M PPD, 5 minutes2. 1.0 M PPD, 5 min3. 1.5 M PPD, 10 min4. 1.5 M PPD + 0.1 M Sucrose 5minutes, load into straw and place in

freezing machine at room temperature.

Can use steps 3 & 4.Recommend 1, 2, 3 & 4.

Freezing of Blastocysts; Fully Expanded Blastocysts (FEB) of good quality

Use QA Blastocyst Freeze Kit ART-8015.

Move FEBs through solutions in following sequence.

1.0 mL/well + oil, 37oC.

1 2

3

Diluent 5 minutes

5% Glycerol, 10 minutes

9% Glycerol/0.2 M Sucrose, 10 minutes.Load into straw and place in freezing machine at room temperature.

Cooling Ramps for Cryopreservation

1. Place cryocontainer (straw) in freezing mchine (Planar) at room temperature.

2. Cool from 22oC to - 7oC at 2oC/minute.

3. Hold for 5 minutes at - 7oC.

4. Seed. Check that seeding is progressing after 3-4 minutes.

5. Hold for 10 minutes at - 7oC after seeding.

6. Cool at 0.3oC/minute to -35oC.

7. Transfer to liquid nitrogen.

Thawing of Embryos

Applies to zygotes, cleaving embryos and blastocysts.

Use QA Thaw Kit ART-8016.Thawing:

Straw: Hold in air for 30 seconds, then agitate in a water bath at 35-37oC until ice has melted.

Vial: Agitate in a water bath at 35-37oC until ice has melted.

Use 35 mm dishes with 2.5 – 3.0 mL of solution/dish, covered with oil.

Empty cryocontainer contents into a dry dish

0.5 M Sucrose37oC10 minutes

0.2 M Sucrose37oC10 minutes

60 mm dish.100 uL drops of Diluent, covered with oil.37oC1 minute in each drop, pipetting back and forth.

ET or culture

COOLING PROTOCOL Sage DFU

1.Embryo to Freezing Medium, 20 min, RToC

2. Load into straw or vial. Cool to -7oC at 2oC/min.

3. Hold at seeding temp for 5 min, seed, hold for another 10 min then cool at about 0.3ºC/min to around –35ºC and then transferred to LN2.

THAWING PROTOCOL ISage DFU

1. Hold straw in air, 30-40 secs

2. Water bath, 30-35oC, until ice melted

3. Transfer container contents to a dry dish and find embryo(s)

4. 0.5 M sucrose, 10 min, RToC

5. 0.2 M sucrose, 10 min, RToC

All at room temperature

THAWING PROTOCOL IISage DFU

6. W1 = Wash Medium, 37oC, 5 min

7. W2 = Wash Medium, 37oC, 5 min

8. 3 x 30 uL equilibrated Culture Medium

All at 37oC

Results from Conventional Human Blastocyst Freezing

Menezo et al, 2001

# Thawing cycles 943

# Transfer cycles 857 (91%)

# Thawed blastocysts 1,993

# Transferred blastocysts 1,596 (80%)

# Clin preg/ET 204 (24%)

# Ongoing preg/ET 171 (20%)

Clinical IR 14.6%

Ongoing IR 12.5%

Live births/frozen blast. 10%

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

Routine Freezing of Blastocysts

• Original blastocyst cryopreservation protocol: modified by Menezo & Veiga (1997) and it became extremely convenient.

• Differing clinics have had inconsistent results with this protocol.

• Much of this has probably been due to inexperience on the part of many embryologists, both with selecting blastocysts of sufficient quality to freeze, and also understanding the subtleties of the cryopreservation technique.

• The most common practice to attempt improved consistency has been to reintroduce one or two glycerol concentration steps in the thaw, with one or two extra sucrose dilutions.

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

Selection Criteria for Human Blastocysts for Cryopreservation:

• Expanding blastocyst growth rate: day-5 > day-6 > day-7• Overall cell number > 60 cells (depending on day of

development)• Relative cell allocation to trophectoderm / inner cell mass• Original quality of early stage embryo: pronuclear

formation, blastomere regularity and mono-nucleation, fragmentation

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

Shady Grove Fertility, Maryland: Cryopreservation Outcomes for 2000 (to June)

Zygote Cleavage Stage Blastocyst Total Thaw 10 10 58 78

Transfer 9 8* 54 71 Viable Pregnancy 2 5 18 25 %/Thaw 20% 50% 31% 32% %/Transfer 22% 62.5% 33% 35%

* thaw at the cleavage stage with culture and transfer at the blastocyst stage.

COMPARISON OF BLASTOCYST CRYOPRESERVATIONSLOW COOLING VERSUS VITRIFICATION (Cryotop)

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

Figure 1. Hatching Blastocyst Post-Thaw.

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

Routine Freezing of Blastocysts

• Original blastocyst cryopreservation protocol: modified by Menezo & Veiga (1997) and it became extremely convenient.

• Differing clinics have had inconsistent results with this protocol.

• Much of this has probably been due to inexperience on the part of many embryologists, both with selecting blastocysts of sufficient quality to freeze, and also understanding the subtleties of the cryopreservation technique.

• The most common practice to attempt improved consistency has been to reintroduce one or two glycerol concentration steps in the thaw, with one or two extra sucrose dilutions.

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

BLASTOCYST CRYOPRESERVATION

FREEZE

1. Holding Medium: modified HTF + 20% HSA.2. Freeze expanding, fully-expanded, and/or hatched or hatching

blastocysts on Day-5/6 (unless fertilization delayed).3. Embryos into modified HTF + 20%HSA @ 37oC, then move onto cool

bench (22oC), and wash through several droplets for about 1 to 2 min.4. Move into 5% glycerol for 8 min.5. 10% glycerol + 0.2M sucrose for 8 min (including loading time). Load

straws / cryo-vials. (1.2ml Nunc cryo-vials containing 0.3ml medium after rinsing).

6. Cool @ –2oC/min to –7.0oC; hold for 15min; “seed” after 5min; -0.3oC/min to –38oC, then plunge into liquid nitrogen for storage.

Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116

BLASTOCYST CRYOPRESERVATION

THAW

1. Room temperature for 1min. Water-bath @ 30oC till ice gone.2. Locate blastocyst in 10% glycerol + 0.4M sucrose for 30 to 40sec.3. 5% glycerol + 0.4M Sucrose for 3min.4. 2.5% glycerol + 0.4M Sucrose for 3min.5. 0.4M Sucrose alone for 3min; move dish to warm microscope/bench.

6. 0.2M Sucrose for 3min.7. 0.1M Sucrose for 3min8. Modified HTF + 20% HSA @ 37oC for three washes, then into culture of HTF + 15% HSA.9. Undertake assisted hatching while blastocyst still collapsed post-thaw.10. Culture for a minimum of 4hrs to observe re-expansion.

Thawing of Blastocysts

More gentle removal of CPAs.For example, Dean Morbeck:

1.

Reagents:

1. Quinn’s Advantage Blastocyst Freeze Solution (9% Glycerol, 0.2M Sucrose; ART-8011; part of Blastocyst Freeze Kit ART-8015)

2. Quinn’s Advantage Embryo Thaw Solution (0.5M Sucrose: ART-8005)

3. Quinn’s Advantage Embryo Thaw Solution (0.2M Sucrose: ART-8007)

4. Quinn’s Advantage Embryo Thaw Solution (Diluent)

Products 2,3 & 4 are in the Embryo thaw kit ART-8016.

Solutions # Diluent 0.2 M Sucrose 0.5 M Sucrose 9% Glycerol, 0.2M Sucrose

1 0 0 1 mL 1 mL

2 0 1 mL 1 mL 0

3 0 1 mL 0 0

4 1 mL 1 mL 0 0

5 1 mL 0 0 0

Thawing of Blastocysts

Dean Morbeck 2.

Make a separate dish for each solution. Make 2 x 200 ul drops each of solutions 1-4 and cover with 8-10 ml oil. For solution 5, make 8-12 x 25-50 ul drops and cover with oil.

Using the 300 um stripper, transfer blastocysts to a 200 ul drop of solution #1 under oil (4.5% glycerol, 0.35M sucrose) at room temperature. Leave blastocysts in solution #1 for 4 minutes and transfer to subsequent solutions according to the following protocol:

Use a new, rinsed 300 um Stripper tip to rinse blastocysts through 6 drops of equilibrated blastocyst media under oil. Place blastocysts in the drops designated for culture.

View blastocysts and return dish to incubator.

Solutions # Time (minutes) Sucrose Glycerol

1 4 0.35 M 4.5%

2 3 0.35 M 0

3 2 0.2 M 0

4 1 0.1 M 0

5 Rinse through drops

0 0

Sage DFU: possible variationsHEPES-HTF w/12 mg/mL HSA

SAME

Diluent 5 min 37oC

5% Gly 10 min 37oC

10% Gly/0.2M Suc 10 min 37oC

   

30-35oC to -6oC @ 2oC/min

SAME2 min seed + 8-13 min total=10-15 min

-7oC to -35oC @ 0.3oC/min

-35oC to -180oC @ 50oC/min

or direct to LN from -35oC

   

straw to RT 20-30 secs SAME; then variation of Tucker dilution protocolwater @ 30-35oC 30 secs

0.5M Suc 5 min 37oC 9% Gly + 0.2M suc, RT 30-40 secs

0.2M Suc 5 min 37oC 4.5% gly + 0.2 suc, 3min (1:1 9% G+0.2 M suc: 0.2 M suc)

Wash 7 x 100 uL HEPES 2.3% gly + 0.2 suc, 3min (1:1 4.5% G+0.2 M suc: 0.2 M suc)

1 min/drop @ 37oCT0.2 M suc, 3 min, move dish to 37oC: 0.1 M suc, 3 min, x3 wash in diluent

transfer to culture medium transfer to culture medium

Freeze

Cool

Thaw

Wade et al., ASRM (2005)Action Rodney Wade

FREEZING  

base medium SAGE products (& uses 0.5 mL vials)

step 1 Place culture dish @ RT 5 min

step 2 5% Gly 10 min RT

step 3 9% Gly/0.2M Suc 10 min RT

   

COOLING  

ramp 1 RT to -7 C @ 2 C/min

Hold 5 min, then seed; hold another 15 min

ramp 3 -7 C to -35 C @ 0.3 C/min

ramp 4  

ramp 5 to LN

   

THAWING ampule held at RT 60 secs

use 2.5 mL of each solution in 35 mm dish overlaid with 1.5 mL of oil

water @ 37 C 2 min

0.5M Suc 5 min RT

0.2M Suc 10 min RT

HEPES 10 min RT to 37 C

   

   

  transfer to culture medium

Wade et al., ASRM (2005)

ROOM TEMP AIR

WATERBATH 37° 0.5M SUCROSE 0.2 SUCROSE M-HTF to 37° IN CULTURE

GROUP 1 1 Minute 2 – 3 Minutes 10 Minutes 10 Minutes 15 Minutes 4 – 8 Hours

GROUP 2 1 Minute 2 – 3 Minutes 5 Minutes 10 Minutes 15 Minutes 4 – 8 Hours

# CPET # TRANSFERS # EMBRYOS THAWED

# EMBRYOSTRANSFERRED

+ PREGNANCY FHT

GROUP 1 123 108 (87.8%) 470 269 (59.23%) 43 (39.8%) 37 (34.26%)

GROUP 2 141 126 (89.4%) 529 277 (52.4%) 83 (65.9%) 62 (49.2%)

P=.02 P=.001 P=0.025

THAW PROCEDURES

Wade et al., ASRM (2005)

The Two Biggest Factors in Slow Freezing v. Vitrification

1. Success rates

2. Time of procedure

COMPARISON OF BLASTOCYST CRYOPRESERVATIONSLOW COOLING VERSUS VITRIFICATION (Cryotop)

Timing of Slow Cooling vs. Vitrification

oC

+20

0

-20

-40

-60

Time minutes

30 60 90 120

Slow coolingVitrification

Timing of Slow Cooling vs. Vitrification

Event Slow Cooling Vitrification

(10 oocytes)

Strip cumuluss

5-10 min

Equilibrate in CPA 20 min 5 + 1 = 6 min

Load device straw/vial 2-3 min eg OPS 15 secs.

Cool to seeding To 15 min

Seed 2 min

Plunge in LN2 & store 5 min

HANDS ON TIME 20-30 min for all 20 oocytes

10 min per group 1-2 oocytes: X5= 50 min

Several patients Can be done together

Each has to be done individually

Final Word

From Lynette ScottFertility Center of New England, Boston

I am still not convinced about Vit at the cost…. Patrick, kits are way over conventional and straw systems prohibitive if you are doing single embryo cryo, which I am as I do single embryo ET and FET in 20-30% of cases.

And if my conventional FET rates = fresh rates, even on IR, why would I increase my cost…… Devils advocate.

Give me a really good reason because time is not one, I am freezing 1-5 patients, 2-10 embryos at a time and with conventional cryo my embryologists are multi- taskers, with vit Forget It!! They are it for a few hours. And you have to have a very “nice” system. Or a very “good” embryologist. I can teach even junior techs to freeze-thaw in conventional systems.  As my FET system works so well, 90% survival, FHB as good as fresh, what are my incentives to change??

Final Word

My personal opinion

1. For both systems, need to optimize culture system, choose the best embryos, and optimize cryopreservation system.

2. There is a place for both systems:

i. Vitrification for oocytes, and in cases where there are only a few embryos and/or 1 or 2 patients.

ii. Slow freezing where there are many embryos and/or patients.