Tissue Culture Series #4: Cell Freezing

Chill freezing media (either commercial cryostar or homemade 90:10 serum:DMSO mixture) prior to use to minimize cell exposure to DMSO. Pre-label cryo storage tubes with cell type, passage number, freeze date, and cell concentration.

Set up a freezing container with isopropanol to enable controlled temperature reduction (1°C/minute). For adherent cells, detach from flask using trypsin, deactivate with media, and transfer to a 15mL conical tube.

Take a small aliquot and perform cell count and viability assessment using a scepter cell counter or hemocytometer. Ensure cells are in exponential growth phase and achieve greater than 90% viability.

Pellet cells by centrifugation and carefully aspirate media without disturbing the pellet. Resuspend cells in chilled freezing media at optimal concentration of 1-2 million cells per milliliter.

Transfer one milliliter aliquots of cell suspension into pre-labeled cryovials, distributing evenly across all labeled tubes.

Place cryovials in the freezing container and transfer to a -80°C freezer overnight to achieve slow, steady temperature reduction that minimizes cell damage.

The next day, quickly transfer the frozen cryovials from the -80°C freezer to liquid nitrogen for permanent long-term storage.