APOPTOSIS IN CHO CELL BATCH CULTURES - EXAMINATION BY FLOW-CYTOMETRY

Chinese hamster ovary cells grown under conditions which are optimal f or the production of a genetically engineered protein in batch culture , lose significant viability shortly after entering the stationary pha se. This cell death was investigated morphologically and was found to be almost exclusively via apoptosis. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidi um iodide. Apoptotic cells could be detected by this method, and by th e radioactive end-labeling of extracted DNA, on all days of culture fr om day 1 to day 7; however, the degree of apoptotic cell death increas ed dramatically when the cells entered the stationary phase, rising to 50-60% of the total cell number at the termination of the culture. Fl ow cytometric analysis showed that the majority of cells underwent apo ptosis whilst in G(1)/G(0) and formed an apoptotic population with hig h DNA FITC end-labeling and hypodiploid propidium iodide binding. Addi tionally, the ability or inability to secrete specific protein product s did not appear to interfere with the development of the apoptotic po pulation with time.