Overview:
All early stage (2PN, Day 2, and Day 3) pre-implantation embryos are frozen in vials with 0.3 ml of freezing media containing 1.5 M 1,2 propanediol and 0.1 M sucrose according to the method of Testart et al. (1986). GlobalART uses the Medicult Freezing and Thawing System in the laboratory.
Below is the protocol from GlobalART USA. Since many laboratories use a similar system to freeze and thaw early stage embryos, they may wish to follow their specific thawing methodologies.
2PN, Day 2 and Day 3 Embryo Thawing Protocol:
Cryoprotectant is removed and the cells rehydrated. Sucrose is included in the thawing solution because of its large MW, making it impermeable to the plasma membrane and aiding in the removal of the cryoprotectant through diffusion.
Reagents:
- 70 percent alcohol
- Embryo Thawing Pack (MediCult, Cat No 1098 4060)
- P-1 Medium (Irvine Scientific, Cat No IR 99242-100)
- Synthetic Serum Substitute (Irvine Scientific, Cat No IR 99193S-1212-1)
Directions:
- Verify name, consent for embryo thawing, and number of embryos to be thawed and transferred (more embryos will have to be thawed if the patient desires a Blastocyst transfer)
- Fill out paper work
- Arrange needed materials
- Wash workspace with 70 percent alcohol
- Prepare embryo thawing plate:
- One plate per patient
- Vial 1: Embryo freezing medium, propanediol, and sucrose-Well 1 and 2
- Vial 2: Embryo freezing medium, propanediol, and sucrose-Well 3
- Vial 3: Embryo freezing medium and sucrose-Well 4
- Vial 4: Embryo freezing medium-Well 5
- Label each plate with the patients' name and number of embryos in the plate.
- Bring solutions to equilibrate to room temperature.
- Remove the cryovials containing the embryos from the liquid nitrogen and hold in air for two minutes.
- Place in a room temperature bath for two minutes. Remove and carefully wipe.
- Remove the solution containing the embryos from cryovials into sterile petri dish.
- Identify the embryo and place into Well 1 and immediately into Well 2 for five minutes. Be as a quick as possible. In the event the embryo is not found, flush the cryovials until the embryo is recovered.
- Gently move the embryos into Well 3 for five minutes. Remember that these embryos are osmotically STRESSED and need to be treated VERY CAREFULLY.
- Move the embryos into Well 4 for 10 minutes. The embryos will look stressed and the membranes will be very fragile.
- Move the embryos into Well 5 for 10 minutes.
- Wash embryos in growth media (P1 + 10%SSS) and place embryos into growth droplets, allowing them to culture/incubate.
Testart, J. et.al., (1986) High pregnancy rate after early human embryo freezing. Fertil Steril 2: 268-272.
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