Possible limitation of growth in human fibroblast cultures by diffusion

Secondary cultures of human diploid fibroblasts which demonstrated density dependent inhibition of cell growth (DDI) were used to study the possible limitation of growth in cell cultures by diffusion. An oscillating platform system is described which insures constant mixing of the medium during the culturing period. Using this system, it was found that a greater number of cells in density inhibited cultures, grown to confluence for four days after initial seeding, could be stimulated to resume growth by a fresh medium change if the cultures were incubated on the oscillating platform than if the cultures were left undisturbed. This greater stimulation on the platform was probably not due to mechanical alterations on the surface of the cells due to motion of the medium as judged by TCA precipitable material released into the medium from cells prelabeled with glucosamine-3H. In spite of this greater stimulation after a single treatment with fresh medium, refeeding the cells on the platform every other day was a 12-day period did not affect the final saturation density achieved in the cultures. The results indicate that diffusion limitation of growth might occur under certain circumstances but that it cannot account entirely for the phenomenon of DDI.