3.2 Freezing Cell
Monolayers
See schematic diagram presented in Fig. 2.
1. Turn on the methanol bath, set the temperature for À5
C, and
make sure the mixer is spinning (stirrer speed at setting 70).
Monitor the temperature of the bath and inside a surrogate
glass vial containing 200 μL medium using calibrated T-type
thermocouples and an OMB-DAQ-55 data acquisition
module.
2. Prepare 10% (w/w) DMSO, 12% (w/w) HES, and 4% (w/w)
chondroitin sulfate (CS) in media (see Note 4).
3. Put 95 μL of EGM-2 in a 15 Â 45 mm glass vial. Using finepoint forceps, transfer a coverslip from each well and place cell
side up in each vial (see Note 5).
Fig. 2 Schematic outline of freezing cell monolayers cultured on fibronectincoated Rinzl coverslips. Coverslips in glass vials are equilibrated at the ice
nucleation temperature (À5
C) in a methanol bath. Extracellular ice formation
is induced using pre-cooled forceps and the monolayer cooled at 0.2
C/min or
1
C/min to À45
C, and then plunged into liquid nitrogen for storage
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Leah A. Marquez-Curtis et al.