Study of the Growth Curve of Vero Cell with Statistical Counting Method Based on the SEM Images

Abstract

For industrial-scale culture of anchorage-dependent mammalian cell, such as vero cell, growth on the microcarriers offers certain advantages. As previous research reported, growth process can be illustrated by a growth curve - cell density versus time, which shows the change of cell density through whole growth process including: first adhesion stage; spreading and division stage; and last confluent stage. The growth curves are controlled by the culture condition (culture medium, temperature, pH value, inoculation concentration, etc.) and space condition (kind of microcarrier, microcarrier size, and surface property, etc.). Therefore, the cell growth curve is not only the important basis for studying growth kinetics, but also for designing biological reactors.

Traditionally, total cell concentration for the microcarrier cultures were determined using the crystal violet method, and the cell nuclei were counted with a hemacytometer. in this method, the cells attached on each microcarrier were trypsinized and dyed by crystal violet dye, so that the number of nuclei counted was statistically representative. in this work, the cells and microcarriers were examined by using an environmental SEM - KYKY1500. As a result, the moist cells and microcarriers in SEM images show their natural characteristics, because this environmental SEM permits biological sample to be observed directly in an environmental specimen chamber without normal preparation for biological sample. As for the normal preparation, it needs a long duration, e.g. 2 days normally, to do fixing, desiccating and gold-coating. in contrast, the operation for observation of the biological samples in this SEM is much simpler. in view of this, a statistical counting method based on the SEM images for determining cell concentration during microcarrier culture was developed.