Serum/Protein-Free Cell Freezing Medium

1. Cryopreserving Cells

For optimum results, cells should be in logarithmic growth phase at the time of freezing.

1.1. For adherent cells, wash with sterile PBS twice and gently detach cells from the substrate using trypsin. Resuspend cells in complete medium. During digestion, carefully handle the adherent cells to avoid cell damage. For suspension cells, proceed directly to step 1.2.

1.2. Obtain a cell suspension using a cell-specific protocol and centrifuge cells for 3-5 minutes at 500 g at 4°C, carefully aspirate the supernatant.

1.3. Determine the viable cell density and percent viability and calculate the required volume of MCE Serum/Protein-Free Cell Freezing Medium to give a final cell density of 1 × 10⁶-10⁷ cells/mL. The whole process is operated on ice to avoid damage to the cells by the protective agent.

1.4. Dispense aliquots of cell suspension into cryovials.

1.5. Directly place the cryovials containing the cell suspension in -80°C refrigerator, and move into liquid nitrogen for long-term storage after 24 h.

2. Thawing Cells

Before starting, warm the required amount of complete medium to 37°C.

2.1. Remove the cryovial from cryo-storage and rapidly thaw it in a 37°C water bath. Thaw sample with gentle swirling until all visible ice has melted.

2.2. Wipe down the outside of cryovials with 75% ethanol and add, dropwise, the appropriate pre-warmed complete medium to suspend cells. Transfer cell suspension to a centrifuge tube that already contains 5-10 mL of complete medium. Ensure complete mixing with regular gentle swirling.

2.3. Centrifuge cells for 3-5 minutes at 500 g at 4°C, carefully aspirate the supernatant.

2.4. Gently resuspend cell pellet in an appropriate volume of pre-warmed complete medium. Transfer cell suspension to sterile culture vessel and place into the recommended culture environment after microscopy.