Cryopreservation of Mammalian Cells in a Mr. Frosty Cell Freezing Container v1

This protocol describes how to cryopreserve adherent cell lines by preparing serum-containing or serum-free freezing media (with DMSO as the cryoprotectant), freezing cells at a controlled rate using a Mr. Frosty container, and storing long-term in liquid nitrogen or a −150 °C freezer. It also outlines a rapid thaw procedure that dilutes and removes DMSO by centrifugation before resuspending and plating cells in fresh culture medium. Expected results are high post-thaw viability and successful reattachment/recovery of cells when DMSO is pre-mixed into freezing media and cells are handled quickly during freezing and thawing. Proper implementation should yield reproducible recovery of healthy, proliferating cultures (including drug-maintained conditions such as VCaP16 frozen with Enzalutamide), with minimal freezing-related stress or loss of cell number.