Cryopreservation and thawing of cells.

Successful cryopreservation of cells requires not only that the cells be handled in a proper fashion for harvesting with equipment in place to ensure consistency, reproducibility, and sterility, but also that a correct choice and amount of cryoprotective agent is added. In general, a controlled freezing rate of 1°C/min is necessary to retain optimal viability of the recovered cells. There are many variations of cell freezing methods in use, including costly electronically regulated control rate freezers, unstandardized, passive isopropyl alcohol freezing containers, and crude rudimentary devices constructed from Styrofoam boxes or paper insulation. However, for the freezing and recovery of cell lines, primary cells, and stem cell cultures, the protocol described in this unit is simple, reproducible, and successful. Not only does it eliminate the need for isopropanol, as well as the costs and hazards associated with its use and disposal, but it provides a uniform method with improved cell viability and recovery.