Protocol for
Cryopreserving Cultured Cells
The
following protocol describes a general procedure for cryopreserving cultured
cells. For detailed protocols, always refer to the cell-specific product
insert.
1.
Prepare freezing medium and store at 2oC
to 8oC until use. Note that the appropriate freezing medium depends
on the cell line.
2.
For adherent cells, gently detach cells
from the tissue culture vessel following the procedure used during the
subculture. Resuspend the cells in complete medium required for that cell type.
Determine
the total number of cells and percent viability using a hemacytometer, cell
counter and Trypan Blue exclusion, or the Countess, Automated Cell Counter.
According to the desiredviable cell density, calculate the required volume of
freezing medium.
4. Centrifuge the cell suspension at approximately 100–200 g for 5 to 10 minutes; aseptically decant supernatant without disturbing the cell pellet.
Note: Centrifugation speed and duration
varies depending on the cell type.
5.
Resuspend the cell pellet in cold
freezing medium at the recommended viable cell density for the specific cell
type.
6.
Dispense aliquots of the cell suspension
into cryogenic storage vials. As you aliquot them, frequently and gently mix
the cells to maintain a homogeneous cell suspension.
7.
Freeze the cells in a controlled rate
freezing apparatus, decreasing the temperature approximately 1oC per
minute. Alternatively, place the cryovials containing the cells in an
isopropanol chamber and store them at –80oC overnight.
8.
Transfer frozen cells to liquid
nitrogen, and store them in the gas phase above the liquid nitrogen.
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