THE LIMITED GROWTH SPAN OF CELL STRAINS ISOLATED FROM THE CHICK EMBRYO

Fibroblasts isolated from embryonic chick skin and muscle have been studied with respect to the following properties, using standard cell culture techniques and measurement of metabolite flux as well as quantitative biochemical assay: (1) Under our culture conditions these cells can undergo a maximum of 25 divisions during a culture period of about 2 months. (2) Cells isolated from 10-12-day-old embryos did not differ significantly in growthspan from cells obtained from 18-20-day-old embryos. (3) A marked decrease in the number of cells present at confluency occurs between the early and late stages of culture. This decrease amounts to about 3.5 per cent per generation if we assume a linear decrease in cell number. (4) The amount of DNA per cell decreases slightly at late culture stages while the ratio of RNA to DNA and of protein to DNA remains essentially constant. The lipid content per DNA appears to increase significantly. (5) The influx of glycine, leucine, and glucose, and the efflux of radioactivity from cells grown in medium containing C-14-glycine was studied, but no significant differences were observed as a function of passage number. (6) Evidence is presented indicating that the inoculation density is an important determinant of division limitation and survival. An hypothesis explaining the phenomenon of growth limitation is advanced which is consistent with this finding and with the observations that feeder cell systems or conditioned medium increase the growth span of euploid cells in vitro.