Low cell viability due to the freezing or thawing of cancer cell lines

Freeze slow, thaw fast.  The process of freezing and thawing cancer cell lines requires extreme attention to protocol.  For freezing, a good freezing media that contains DMSO is added to the correct number of cells following the manufacturer’s instructions.  The vital part of the freezing process is to ensur that the freezing process is slow.  Typically, the prepared cells are placed inside of commercially available freezing chambers that slowly freeze the cells at a rate of 1°C per minute in a -80°C freezer.  After completely frozen, the vials need to be stored in the vapor phase of a liquid nitrogen storage tank.

*Tip from the Bench: If a freezing vial is not available, a common trick to freezing cultured cells is to place the vial containing cells and freezing media inside of a Styrofoam shipper at room temperature, and then place the entire box into a -80°C freezer overnight.

The thawing process for cultured cancer cell lines is the exact opposite of the freezing process.  Thawing frozen cultured cells needs to happen very quickly (i.e. less than 1 minutes) to maintain high cell viability.  Typically this is accomplished by placing the frozen vial in a 37°C water bath.

*Tip from the Bench:  Thawed cells are extremely fragile.  It is recommended to add the thawed cells to warm media and culture overnight.  Thus, unlike previous practices, do not manipulate, centrifuge or wash the cells until the next day.

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