(f) Transfer the sterile-filtrated solution into a 15 mL Corning Falcon High-Clarity Polypropylene Centrifuge Tube.
(g) The remaining 20 mL DPBS/HSA are needed as washing
solutions (2x10 mL). These 2 Â 10 mL solutions should
be sterile filtrated in the same way like the thawing
solutions.
(h) Keep the sterile-filtrated thawing and washing solutions in
the refrigerator until the thawing procedure will be performed, no later than the following day (see Note 4).
3.5.2 Thawing
and Removal
of Cryoprotectants,
Restoration of Cell
Volumes, Washing,
and Equilibration
1. Before starting the thawing process: Transfer the preprepared
sucrose/wash solutions into a six-well plate according to Fig. 4.
2. Transport the cryo vials to be thawed from the storage place in
the cryobank in liquid nitrogen (Dewar transport container) to
the laboratory.
3. Remove the cryo vials from liquid nitrogen/transport
container.
4. Preincubate the cryo vials with frozen ovarian cortex pieces at
room temperature for approximately 30 s.
5. Transfer the pre-thawed cryo vials into a 37
C warm water
bath for ~120 s. Use sterile/autoclaved water for filling the
water bath.
6. While continuously observing the frozen medium in the cryo
vials, remove the cryo vials from the water bath as soon as the
medium has liquefied, and only a central ice crystal of the
frozen medium is visible (the temperature in the cryo vials is
then ~4–10
C).
10mL
0,75M
15 min
10mL
0,375M
15 min
10mL
0,125M
15 min
10mL
DPBS/HSA
10 min
5mL
DPBS
5 min
Fig. 4 Sucrose/wash solutions prepared in a six-well plate
494
Jana Liebenthron and Markus Montag