EMBRYO CRYOPRESERVATION

CRYOPRESERVATION TECHNIQUE (Clinic and research center Dr. Roger Abdelmasih)

Oocytes, pronuclear stage to 8 cell embryos

Equipment

Freezing machine: Planer Kryo 10 – Series II liquid Nitrogen (LN2) bottle:must be used exclusively for human embryos or oocytes. Straws or tubes for freezing: they need to be sterile, non-toxic and high quality. Pinças: for seeding performance.

Freezing and thawing solutions

FREEZE – KIT 1/ THAW – KIT 1 containing 1,2 propandiol (Scandinavian IVF Science )

Freezing program

Freezing procedure time includes equilibration of cryoprotectants during approximately 2 hours.

Initial temperature: + 18.0°C

1st step: - 2.0°C / min until -7.0°C

2nd step: wait at - 7.0oC for 10 min, SEEDING after 2 minutes

3rd step: - 0.3oC / min until - 30oC

4th step: -30°C lowering down to -80°C ( minimum 10°C / min )

Remove straws and place directly into LN2, store submerged into LN2 (bottle).

Freeze – Kit1 (components)

Cryo PBS = Buffer phosphate solution with 25mg/ml human serum albumine (HSA)

Freezing solution 1 = FS1 = 1.5M PrOH in Cryo-PBS

Freezing solution 2 = FS2 = 1.5M PrOH + 0.1M sucrose in Cryo-PBS

ESSENTIAL POINTS FOR A GOOD FREEZING

Cryopreserve only high quality embryos, since survival rates after thawing depends on the initial embryo quality.

Check complete patient’s name and prepare all terms of consent before beginning the procedure.

Procedures

1 - Prepare the dishes for the freezing solutions, pipette apropriate Cryo-PBS, FS1 and FS2 volumes inside the respective dishes. Equilibrate room temperature.

2 - Place embryos for freezing in Cryo-PBS, observe morphology and write down in detail (2-5 minutes).

3 - Gently place embryos for freezing in FS1 for 10 minutes (never leave them over 20 minutes). The embryo cells will shrink and then reequilibrate during this step.

4 - Move embryos to FS2 and then place them inside the straws with a 1 ml syringe connected to the straw (the syringes are connected to the straws using a 1 cm plastic tube) Remove the straws and close, avoiding liquid nitrogen to get in.

5 - Place inside the freezing machine at room temperature and begin the freezing program.

6- Make the manual seeding in the straws at – 7oC with a pinça, previously embedded in LN2, near the cotton plug. Do not make the seeding ner the embryos. Do not let straws fall or balançar. Air bubbles inside the straws may reduce cell survival rate. Continue freezing program.

7 - Freezing takes approximately two hours. Special care must be taken when manipulaing palhets at low temperatures because they can unfreeze easily. Place inside liquid nitrogen and store at – 196oC.

THAWING PROGRAM

THAW – KIT 1 (components)

Thawing solution 1 = TS1 = 1.0 M PrOH + 0.2 Sucrose in Cryo-PBS

Thawing solution 2 = TS2 = 0.5 M PrOH + 0.2 Sucrose in Cryo-PBS

Thawing solution 3 = TS3 = 0.2 M PrOH + 0.2 Sucrose in Cryo-PBS

Essential points for a good thaw:

Thaw one straw at a time.

Perform all steps at room temperature (approximately +18 to + 20oC).

Check patient’s complete name and prepare all documentation.

Procedure

1 - Identify patient and find straw. Prepare all working paper. Leave straw above LN2 on a small grade until total thawing. Prepare dishes for thawing solutions and pipette appropriate TS1, TS2, TS3 and Cryo-PBS quantities.

2 - Remove straw and thaw it in the air for 30 seconds. During this period hold straw carefully and examine it to look for air bubble formation or ruptures in the straw closing.

3 - Place in Mary’s Bath at + 30oC for 30 seconds. Remove and dry carefully. Cut one of the straw extremities with sterile scissors and connect to a 1 ml syringe. Cut the other extremitie carefully, without moving the straw (holding the straw tight) and without making air bubbles.

4 - Gently place embryos inside TS1. Observe embryos coming out of the straw and, if you cannot se all of them, quickly reload the straw and wash gently. Occasionally embryos might stick to the straw extremities.

5 - Incubate embryos for 5 minutes in TS1.

6 - Gently move embryos from TS2 for 5 minutes (approximetely). Remember these embryos are osmotically stressed and require special care in handling.

7 - Move embryos to TS3 for 5-10 minutes. Embryos will be stressed and membranes very fragile.

8 - Place embryos in Cryo_PBS at room temperature for about 10 minutes and then move them to a recipiente pre-heated to + 37oC (heater platinum). Do not put them in the incubator with CO2 because Cryo-PBS buffering capacity is not enough for the 5% co2.

9 - Embryos may be placed inside the IVF 50, G1.2 or G2.2 medium. Perform classification and write down morphology and comparative survival with pre-freezing. Embryos may be immediately transfered or left in culture. Oocytes in pronuclear stage must stay in culture for all night in IVF-50 or G1.1 medium and only if normal cleavage has ocurred may they be tranferred.

10- The thaw-cycle is very important for the success of embryo cryopreservation. Make sure the embryos were tranfered to teh uterus on the appropriate day. Embryos may be transferred in a natural or artificial cycle.

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