Part VI: Freezing and viability staining of cells

Welcome to the last of a series of articles providing useful hints for culturing animal cells. This article contains considerations for freezing and viability staining of cells.

Cell freezing

For some cell cultures, especially valuable ones, it is common practice to maintain a two-tiered frozen cell bank: a master and a working cell bank. The working cell bank comprises cells from one of the master bank samples, which have been grown for several passages before storage. If and when future cell samples are needed, they are taken from the working cell bank. The master cell bank is used only when absolutely necessary. This ensures that a stock of cells with a low passage number is maintained, and avoids genetic variation within the culture.

1. Check that cells are healthy, not contaminated, and have the correct morphology.

2. Change the medium 24 h before freezing the cells.

Tip - Adherent and suspension cell cultures should not be at a high density for freezing. We recommend freezing cells when they are in the logarithmic growth phase.

3. Adherent cultures: harvest the cells by trypsinization, resuspend in medium containing serum, centrifuge at 200 x g for 5 min, and then resuspend cells in freezing medium (see Table 1) at a density of 35 x 106 cells/ml.

Suspension cultures: centrifuge the cells at 200 x g for 5 min, and resuspend in freezing medium (see Table 1) at a density of 510 x 106 cells/ml.

IMPORTANT: Freezing medium containing DMSO is hazardous and should be handled with caution.

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