Separation of clonogenic cells from stationary phase cultures by density gradient centrifugation

Abstract

The density and plating efficiency (PE) of Chinese hamster ovary (CHO) cells were monitored from exponential through late stationary phase. During the first 10 days of growth, centrifugation of cells in linear density gradients of either Renografin or bovine serum albumin (BSA) yielded a single band of cells. The PE of cells collected from Renografin gradients decreased with the age of the culture from about 90% in exponential phase to about 52% at day 10. No significant difference in PE was observed between cells spun in Renografin and BSA gradients and control cells plated directly. At late-stationary phase (13 and 14 days after plating), three populations of cells were observed in Renografin gradients at densities of 1.102, 1.148 and >1.199 g/cm 3 (pellet). The average PE for these three fractions of cells were 54, 6 and <1%, respectively, as compared with 21% for control cells plated directly. Analogous results were obtained with BSA gradients. Cells were found to exhibit significant variations in density which were related to the age of the culture. The separation and enrichment of clonogenic cells from stationary phase cultures were achieved using both density gradients of Renografin and of BSA.