Thawing Tissue Culture Cells

Very easy. The goal is to get the frozen cells transitioned to growth temperature and conditions as rapidly as possible.

1. remove the vial from the liquid nitrogen freezer
   
2. in a laminar flow hood, wearing latex gloves, wipe down your gloves and the complete outer surface of the vial with a 70% ethanol solution (to minimize contamination)
   
3. prepare a dish with medium of the same size the cells were initially collected upon for freezing (eg, if the cells were initially collected for freezing from a 10 cm diameter tissue culture plate, thaw the frozen cells back onto a new 10 cm diameter plate). put the dish with media in the incubator to equilibrate temperature and pH
   
   • replating the cells after thawing at either too high or too low a density is not good
     
4. unscrew the vial to equilibrate the gas pressure in the vial with normal atmospheric pressure, then screw the lid back down to seal the vial
   
5. hold the vial in your (latex) gloved hand until it is partially thawed. alternatively: incubate with vigorous agitation in a 37C water bath until completely thawed
   
   • this won't hurt your hand -- the vial doesn't have that much heat capacity in it and will very rapidly warm up once removed from the freezer
     
6. shake the vial until the remaining frozen material in the vial sloshes back and forth against the melted solution in the vial
   
7. remove the cap of the vial and dump the half-frozen cell slurry directly into the medium in the previously prepared plate
   
8. swirl the plate until the remaining frozen material has completely thawed, then immediately place the plate in a humidified tissue culture incubator
   

see also: FreezingTissueCultureCells

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Comments

• in general, do NOT add selective drugs to the medium until 24 hours after thawing