THAWING AND RECOVERING HUMAN CELLS

When cryopreserved cells are needed for study, they should be thawed rapidly and plated
at high density to optimize recovery.
CAUTION: Protective clothing, particularly insulated gloves and goggles, should be worn
when removing frozen vials or ampules from the liquid nitrogen freezer. The room
containing the liquid nitrogen freezer should be well-ventilated. Care should be taken not
to spill liquid nitrogen on the skin.
Additional Materials (also see Basic Protocol)
70% (v/v) ethanol
Complete medium/20% FBS (e.g., supplemented DMEM-20, APPENDIX 2A), 37°C
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2
incubator unless otherwise specified. Some media (e.g., DMEM) may require altered
levels of CO2 to maintain pH 7.4.
1. Remove vial from liquid nitrogen freezer and immediately place it into a 37°C water
bath. Agitate vial continuously until medium is thawed.
The medium usually thaws in <60 sec.
Cells should be thawed as quickly as possible to prevent formation of ice crystals that can
cause cell lysis. Try to avoid getting water around the cap of the vial.
2. Wipe top of vial with 70% ethanol before opening.
Some labs prefer to submerge the vial in 70% ethanol and air dry before opening.
3. Transfer thawed cell suspension into a sterile centrifuge tube containing 2 ml warm
complete medium/20% FBS. Centrifuge 10 min at 150 to 200 × g (∼1000 rpm in
Fisher Centrific), room temperature. Discard supernatant.
Cells are washed with fresh medium to remove residual DMSO.
4. Gently resuspend cell pellet in small amount (∼1 ml) of complete medium/20% FBS
and transfer to properly labeled culture plate containing the appropriate amount of
medium.
Cultures are reestablished at a higher cell density than that used for original cultures
because there is some cell death associated with freezing. Generally, 1 ml of cell suspension
is reseeded in 5 to 20 ml medium.
5. Check cultures after ∼24 hr to ensure that cells have attached to the plate.
6. Change medium after 5 to 7 days or when pH indicator (e.g., phenol red) in medium
changes color. Keep cultures in medium with 20% FBS until cell line is reestablished.
If recovery rate is extremely low, only a subpopulation of the original culture may be
growing; be especially careful of this when working with cell lines known to be mosaic.