Cooperative cell interactions in pure and mixed culture

Abstract

The population growth and cell morphology of mouse A9s and human foreskin fibroblasts (HFFs) were used to investigate cooperative cell interactions in pure and mixed culture. It was found that neither cell line employed the bacterial growth optimization strategy whereby the lag phase is used to build up stockpiles of materials which serve as bottlenecks to limit growth. Instead, a strategy is employed which requires colony formation and cooperative cell interaction. The behavior of cell growth rates as a function of population density and time after seeding is more complicated than generally appreciated. At sparse density, shortly after seeding, there is a period of growth rate acceleration which is dependent upon population density. As moderate densities are achieved, this acceleration ceases and the growth rate levels off at a high value. As the cultures approach confluency, contact inhibition of growth occurs, and the growth rate decelerates to a lower but non-zero value. During non-confluency the growth rate of cultures is inversely proportional to the percent of singlets (individual cells not in contact with other cells) within a population. Moreover, there is a hysteresis-like effect in that the growth properties of cells depend not only upon momentary conditions, but upon the prior history of cell interactions as well. These effects are not the result of medium conditioning. Instead, they appear to involve either direct cell contact or at least the very close proximity of cells. During non-confluency the growth kinetics of A9s and HFFs in mixed culture are independent of one another. At near confluency, the A9s unilaterally inhibit further HFF proliferation, but themselves continue to reproduce past confluency by invading a second niche—the culture fluid overlying the mixed monolayer. Inhibition of HFF growth by A9s involves territorial exclusion, whereby HFFs are excluded from local areas heavily (but non-confluently) populated by A9s. Locally confluent domains of A9s expand by proliferation, and apparently cause nearby HFFs to retreat before them. As this encroachment progresses, HFFs seem to be herded together by the A9s into ribbon-shaped enclaves which cease proliferation but pile up into multi-layers.