Cryoprotectant Solution: 10 ml
Sucrose 860 mg
Bovine Serum Albumin (BSA) 80 mg
M2 medium ~6.5 ml
Dissolve and filter through a 0.22 um Millipore filter.
Add 3.3 ml Dimethyl Sulphoxide while agitating the solution (thisprevents
the solution from getting too hot which may precipitate the BSA).
Adjust the volume to 10 ml (if necessary). Aliquot into sterile
Eppendorf tubes and store at -200C (for 2 to 3 months).
Procedure:
A. Snap Freeze
1.Collect 8 - cell embryos in a minimal volume of M2 using
a drawn-out Pasteur pipet.
2.Transfer embryos into the cryoprotectant solution inside an
artificial insemination straw (30 to 40 ul cryoprotectant solution).
3.Heat seal the end of the straw.
4.Incubate straw on ice for 3 minutes.
5.Immerse the straw directly into liquid nitrogen.
B.Thawing of Embryos
Media:
M2
M16
0.25M sucrose solution (860 mg sucrose in 10 ml M2)
CAUTION: The embryos are fragile and should be handledipipeted with extreme care at each step.
1.Remove straw from liquid nitrogen and wipe with Kimwipe.
Make sure that no liquid nitrogen is left in the straw as this
may force the cyprotectant out of the straw.
2.Immediately immerse the straw in a 35 to 37oC waterbath and
incubate for 3 to 6 seconds or until the ice has melted.
3.Remove the straw and dry with Kimwipe.
4.Cut off the heat sealed end and expel the cryoprotectant solution
into 2 ml of the 0.25M sucrose solution using a 1 ml syringe with
tubing on the end.
5.Incubate for 10 minutes.
6.Wash the embryos very gently through several droplets of M2:
7.Culture overnight in M16~at 370C, 5% CO2.
8.Transfer the blastocysts into the uterus of recipient mice using
M2 as the transfer media.