(58d) Automated Flow Cytometry for Monitoring and Control of Cho Cell Cultures

A significant problem in mammalian cell culture is the direct assessment of the concentration and composition of the biomass. It is well recognized that considerable single-cell variability exists in a cell culture and that this heterogeneity affects culture performance. Such culture variability is also likely to a large extent responsible for variations that are observed in the final product obtained. Therefore, to further understand how culture heterogeneity change, we developed an automated flow cytometry system. Automated flow cytometry can provide online cell number and single-cell data on virtually any characteristics of the sub-populations for which a flow cytometry stain exists.

To demonstrate the utility of this instrumentation we monitored transient gene expression in a CHO cell culture. Previous experimental studies show a characteristic peak in average protein expression per cell after transfection followed by an exponential decrease of the expressed protein. The frequent sampling in this study reveals that after electroporation, the exponential decay of single cell expression of enhanced Green Fluorescent Protein (eGfp) occurs in discrete steps. We attribute this to the absence of plasmid replication and to symmetric partitioning of plasmid and eGfp between dividing cells. This is reflected in the total eGfp concentration, which increased at a constant rate throughout the experiment. Additionally, the data, with a sampling period of 20 minutes, provide a detailed time course of cell physiology during recovery to electroporation.

Moreover, automated flow cytometry can also be used for CHO cell culture control. We show that an increase in the apoptotic sub-population from a steady state 6x10³ cells/mL can predict the onset of stationary phase by approximately 20 hours. Detecting a population this dilute is possible due to the greater sensitivity and reproducibility of automated flow cytometry. Predicting the onset of stationary phase is useful to establish a completely automated culture scale-up process. Alternately, having cell concentration as an online signal enables precise control of a culture based on the exact moment it changes its growth kinetics.

Therefore, the instrumentation represents a useful, new tool for cell culture monitoring and control that was not available to date.