Growth regulation in multilayered cultures of human diploid fibroblasts: the roles of contact, movement and matrix production.

Early subcultures of human embryonic lung fibroblasts are exceptional, as they grow far beyond confluence before growth ceases: the stationary dish may well contain 3-10 monolayer equivalents. Maximal growth rates, however, occur at about one-sixth confluence when doubling times are 15-20 hr; a density at which cell contacts begin to become frequent. The fact that a slowing down of growth is first apparent at such low densities argues against this regulation being due to diffusion effects. Confirmation of the role of short-range or contact interactions in growth regulation comes from an experiment using mixed cultures of fibroblasts: this shows that growth inhibition is not carried by medium-borne influences but depends on short-range (less than 1 mm) interactions. Evidence that cells can escape the effects of such contact interactions and so divide comes from time-lapse studies of dense cultures: there is a burst of motility soon after a fresh-medium change, which is followed by a burst of mitosis approximately 20 hr later. A medium change to conditioned medium supplemented with 10% foetal calf serum leads to neither the burst of motility nor the subsequent burst of mitosis, although this medium is better able to support the growth of sparse cells than is fresh medium. Data are also presented to show that the amount of collagen deposited in superconfluent cultures affects their growth: the stimulation of collagen production with ascorbic acid leads to an unexpectedly low stationary cell density and rather less movement in the culture. This result suggests that the collagen stabilizes cell contacts that are responsible for growth inhibition. The question of why these cells grow more slowly as density increases cannot be answered directly by these experiments; nevertheless, the results suggest that cell contact affects the permeability of the cell membrane to medium.