Cell Counting and Viability Measurements

Abstract

The accurate determination of cell growth is pivotal to monitoring a bioprocess. Direct methods to determine the cells in a bioprocess include microscopic counting, electronic particle counting, biomass monitoring, and image analysis. These methods work most simply when a fixed volume sample can be taken from a suspension culture. Manual microscopic counting is laborious but affords the advantage of allowing cell viability to be determined if a suitable dye is included. Electronic particle counting is a rapid method for replicate samples, but some data distortion may occur if the sample has significant cell debris or cell aggregates. The use of a biomass probe detects cells by the dielectric properties and can be used as a continuous monitor of the progress of a culture. Image analysis based on the use of digital camera images acquired through a microscope has advanced rapidly with the availability of several commercially available software packages.

Indirect methods of cell determination involve the chemical analysis of a culture component or a measure of metabolic activity. These methods are most useful when it is difficult to obtain intact cell samples. However, the relationship between these parameters and the cell number may not be linear through the phases of a cell culture. The determination of nucleic acid (DNA) or total protein can be used as an estimate of biomass, while the depletion of glucose from the media can be used as an estimate of cellular activity. The state of cellular viability may be measured by the release of an enzyme such as lactate dehydrogenase (LDH) or more directly from the intracellular adenylate energy charge from cell lysates. Alternatively, radioactive techniques may be used to for an accurate determination of cellular protein synthesis.