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Chapter: Basic Concept of Biotechnology - Animal Biotechnology

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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.

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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