EMBRYO CRYOPRESERVATION

BLASTOCYST STAGE

Equipment

Freezing machine: Planer Kryo 10 series II

Liquid Nitrogen botijao (LN2): must be used exclusively for human embryos or oocytes.

Freezing straws or tubes need to be sterile, non-toxic and high quality.

Pinças: for "seeding" procedure.

Freezing and thawing solutions

FREEZE – KIT 2/THAW - KIT 2 containing glycerol and sucrose

Freezing program

The freezing procedure duration includes cryoprotectants equilibration, which takes about two hours and a half.

Initial temperature: + 18.0°C

1st step: - 2.0°C / min until -7.0°C

2nd step: wait at -6.8°C for 5 min and perform the manual SEEDING

3rd step: - 0.3°C / min until - 40°C

4th step: - 40°C down to -80°C (at least 10°C / min)

Remove the straws and place them diretly into the LN2, store submerged in LN2 (botijão).

FREEZE – KIT 2 (components)

Freezing solution 1 = FS1 = 5 % glycerol in G2.2 medium with HEPES.

Freezing solution 2 = FS2 = 9 % glycerol + 0.2 M sucrose in G2.2 medium HEPES.

Essential points for a good freezing

Freeze blastocysts at day 5 or 6.

Do not freeze blastocysts if they are not expanded.

Freeze only high quality blastocysts.

Freeze only one blastocyst per straw.

Check complete patient’s name and all other terms of consent before going on to the procedure.

Procedure

1 - Prepare dishes for freezing solutions, pipette appropriate FS1 and FS2 volumes inside their respective dishes. Equilibrate solutions at + 37°C and 5 % co2 for 3 to 4 hours.

2 - Remove dishes from the incubator 5 minutes before they are used. This alows the medium to cool down at room temperature until it reaches the appropriate pH. Freezing solutions contain HEPES and are able to stay at room temperature during this procedure.

3 - Place embryos for freezing inside FS1 (5% glycerol) for 10 minutes. Leave resting at room temperature.

4 - Move embryos to FS2 (9% glycerol) for 10 minutes. Leave them at room temperature and hen place them inside the straws with a 1 ml syringe connected to a straw (syringes are connected to the straws using 1 cm plastic tube). Remove straws and close, avoiding the entrance of liquid nitrogen.

5 - Place it inside the freezing machine at a temperature of + 18°C and begin the freezing program.

6 - Make the manual seeding in the straws at – 6.8°C with a pinça, previously deepened into the LN2, ner the cotton plug. Do not make the seeding near the embryos.Do not let strws shake or fall. If there are air bubbles inside the straws, this may reduce cell survival rates. Continue freezing program.

7 - Pay special attention to straw handling at low temperatures because they can easily unfreeze. Place them inside liquid nitrogen and store at – 196°C.

Thawing program

THAW - KIT 2 ( components )

Thawing solution 1 = TS1 = 0.5 M sucrose in G2.2 medium with HEPES

Thawing solution 2 = TS2 = 0.2 M sucrose in G2.2 medium with HEPES

Essential points for a good thaw

Thaw one straw at a time.

Perform all steps at room temperature (approximately +18 a + 20°C ).

Check complete patient’s name and preparec all documentation.

Blastocysts must be thawed at least 3-4 hours before transference, allowing their complete expansion.

Transfer only reexpanded blastocysts.

Procedure

1 - Identify dishes containing small thawing solutions until complete and pipette appropriate quantities of TS1 and TS2 on the respective dishes. Equilibrate solutions in + 30°C and 5% CO2 for 3 to 4 hours.

2 - Remove dishes from incubator 5 minutes before being used. This will allow the medium to equilibrate at room temperature. Thjawing solutions contain HEPES and may rest at room temperature for a maximum of 20 minutes.

3 - Check if washing dish and culture dish for embryo tranference with G2.2 medium are equiolibrated. Confirm patients name and where he respective straw is. Leave straw above LN2 on a small shelf until total defrost.

4 - Remove straw and thaw it in the air for 30 seconds. During this peiod hold the straw carefully and examine it looking for the formation of air bubbles or breaches at its closing.

5 - Place it in Mary’s Bath at + 30°C for 30 seconds. Remove and carefully dry the excess water. Cut one straw extremity with sterile scissors and connect to a 1 ml syringe. Cut other extremity carefully, do not wavve the straw or make air bubbles.

6 - Gently place blastocysts inside TS1 and incubate for 10 minutes. Observe blastocysts coming out of the straw and, if you cannot see them all, quickly reload the straw and wash gently. Occasionally embryos will stick to the straw sidesleave them at room tempreature for 10 minutes.

7 - Gently move the blastocysts to TS2 for 10 minutes. Remember that these blastocysts are osmotically stresed, and need to be carefully handled. Leave at room temperature.

8 - Wash blastocysts in G2.2 medium and place them inside the tranference medium and incubate them at + 37oC and 5% CO2 for at least 3-4 hours. Transfer only reexpended blastocysts.

9 - The thaw-cycle is very important for the success of embryo cryopreservation. Make sure the embryos were tranferred to the uterus on the appropriate day. Embryos can be tranferred in a natural or artificial cycle.

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