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CryopreservationSlow Freezing
Patrick Quinn PhD, [email protected]
Sage In-vitro Fertilization, Inc
Redmond, Oregon, USA
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DISCLOSURE
I am Vice President of research and Development at Sage In-Vitro Fertilization, Inc.
We produce a range of commercial ART media products
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Sage IVF
Production @
Pasadena
Redmond
Cooper Surgical,Trumbull
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Sage IVF
Southern California
Central Oregon
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Slow-freezing method
Vitrification
Attention to the size
Ice Crystal Ice Crystal FormationFormation
EquilibratiEquilibrationon
SeedingSeeding Slow Slow freezing freezing
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Slow cooling requires a minimum of ~140 minutes.
Greatest problems:
extra & intracellular ice causing fracture osmotic swelling & shrinkingsolution effectchemical toxicity of CPAsexpensive equipment
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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;
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• 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
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Thawing Solutions
• Hypertonic solution is required, i.e. non-permeable sucrose is added to prevent swelling and shrinkage of the cells;
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• Chilling injury;
• Cryoprotectant (toxicity and temperature);
• Osmotic injury;
• Speed of freezing and thawing;
Factors affecting successful frozen-thawing
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Step by Step Use of Sage Embryo Cryopreservation
Media
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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
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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.
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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.
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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.
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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
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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.
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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
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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
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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%
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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.
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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
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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.
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COMPARISON OF BLASTOCYST CRYOPRESERVATIONSLOW COOLING VERSUS VITRIFICATION (Cryotop)
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Tucker (2001) ART Media, AAB CRB 5th Annual Symposium, pp 95-116
Figure 1. Hatching Blastocyst Post-Thaw.
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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.
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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.
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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.
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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
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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
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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
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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
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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
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Wade et al., ASRM (2005)
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The Two Biggest Factors in Slow Freezing v. Vitrification
1. Success rates
2. Time of procedure
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COMPARISON OF BLASTOCYST CRYOPRESERVATIONSLOW COOLING VERSUS VITRIFICATION (Cryotop)
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Timing of Slow Cooling vs. Vitrification
oC
+20
0
-20
-40
-60
Time minutes
30 60 90 120
Slow coolingVitrification
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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
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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??
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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.