Article: Comparing Automated And Manual Cell Counts For Cell Culture Applications

By Arun Tholudur, Luis Giron, Kohinoor Alam, Tiffany Thomas, Eric Garr, Gresham Weatherly, Keith Kulowiec, Melissa Quick, and Scot Shepard

In biomanufacturing based on animal cell culture, cell enumeration and viability determination are often accomplished using the trypan blue dye-exclusion method. This method is based on the fact that viable cells exclude the dye and remain visually clear, whereas nonviable cells are stained blue.

Historically, cell counts using the technique have been performed manually using a hemacytometer. Cell culture samples are mixed (after dilution in an isotonic buffer, if necessary) with known volumes of trypan blue dye, then loaded into a hemacytometer and counted under a microscope (usually at 100× magnification). It has long been recognized that cell counts obtained using this procedure are subject to inter- and intraanalyst variability that arises from subjective assessments and cell staining, sample dilution, and hemacytometer loading. Two parameters that greatly affect the accuracy of cell enumeration are the number of hemacytometer chambers loaded (replicates) and the number of cells counted within each chamber.

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