Resource Manual - International Embryo Transfer Society

Hybrimax, D-2650)) and 0.1 M sucrose (i.e. 1.71g sucrose (e.g. Sigma S-7903)) made up to 50 ml with
physiological, protein free, buffer (e.g. PBS). If filters which tolerate DMSO are available then sterilize
the DMSO+ sucrose solution. If DMSO tolerant filters are not available then filter the sucrose + PBS
and then add the DMSO. If DMSO is not available then 1.5 M ethylene glycol or 1.5 M propane diol can
be used instead. If a different cryoprotectant is used then the dilution solutions should be made up with
the same (corresponding) cryoprotectant.
Prepare at least 10 ml cryoprotectant per ovary
Pre-cool cryovials (e.g. 1.7 ml Nunc conical, star based, internal thread, CRYO tubes Cat #
377224) on ice. It is easiest if these are each pre-filled with 1 ml cryoprotectant.
It is best if at least 2 ml cryoprotectant be prepared for each tube that is frozen (1 ml for the first
equilibration step, a further 1 ml for each freezing ampoule/vial). This can be made up the day before.
Equilibration procedure:
Once tissue has been dissected, placed it in a tube (e.g. 5 or 10 ml Falcon tube) containing pre-cooled
(ice cold) cryoprotectant solution. Every 5-10 minutes agitate the tube so that all pieces become exposed
to the cryoprotectant. After 15-20 min tip the contents of the tube out into a sterile dish and rapidly
allocate tissue to each vial. Distribute the material as evenly as possible between all vials.
Note: label the vials before freezing commences.
Screw the cap back onto each vial and return to the ice. After the tissue has had a total of 30 min on ice
they are ready to be placed in the freezing machine or in the passive cooling device (―Mr Frosty‖).
Passive cooling:
Place the passive cooling container in a –80 freezer or on dry ice. After 12 to 24 h, remove the vials and
place them in liquid nitrogen for storage.
Cryoporeservation in a freezing machine
Ovarian tissue cryopreserved at 0.3C/ min in a biological freezer is better preserved than material
cryopreserved in a passive cooling device (e.g. Mr Frosty). The difference is however not big, and must
be counterbalanced against the detrimental effect of deterioration with time after collection. Thus
material will be of better quality if it is frozen at 1C/ min (Mr Frosty on dry ice) as soon as possible after
collection, than if the cryopreservation is delayed but then cooled at 0.3C/ min (e.g. by being transported
to a site with a biological freezer).
After loading the vials containing ovarian tissue into a freezing machine they should be seeded to initiate
ice formation (vials placed in a ―mr frosty‖ are not seeded). Seeding is usually performed once the vials
have reached –6ºC (this can take 5 minutes). Seed each vial by pulling it up and touching its outer
surface with a large pair of forceps pre-cooled in liquid nitrogen (place the forceps just above the
meniscus of the liquid, taking care to not touch the sample). After all vials have been seeded re-examine
each one in turn to establish that the solution still contains ice crystals.
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