Resuscitation of Frozen Cells

1. Remove one vial from the cell bank in LN2 after 24 h of storage and immediately thaw by immersion in a water bath at 37°C (see Note 26).

2. As the last of the ice disappears, transfer the suspension to a centrifuge tube (see Note 27). Slowly add 10 mL of CM at 4°C to the cells with gentle mixing and centrifuge the suspension at 300g for 10 min at 4°C.

3. Wash the cells by resuspending the pellet in 10 mL of CM at 4°C and then centrifuging as before.

4. Finally resuspend the cells in 1 mL of CM and count as described in Subheading 3.3.1.

5. Adjust the cell concentration to 1 x 106/mL in CM, add to a 25-cm2 flask, and incubate as described in Subheading 3.1.2. (see Note 28).

6. Visually monitor the cells daily for growth and lack of contamination. Expansion of the cells can be taken as evidence of a viable cell bank and also that no microbial contamination of the cells occurred during the freezing/resuscitation process (see Note 29).

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