Cell saturation density is not determined by a diffusion-limited process

Abstract

THE mouse cell line 3T3 ceases cell division at a confluent saturation density despite regular medium replacement. Also, when a confluent quiescent monolayer of 3T3 is wounded, cells migrate into the wound free of cell contact and initiate DNA synthesis, while cells in the monolayer remain arrested¹. As the cells in both areas are in the same bulk medium, this experiment proves that depletion of growth factors in the bulk medium is itself not sufficient to account for density-dependent inhibition of growth. Two hypotheses have been proposed to explain the wounding experiment. The first states that direct cell–cell contact, perhaps involving the interaction of a receptor with its ligand on an adjacent cell, provides a negative signal for growth¹. The second hypothesis states that growth ceases at a confluent cell density because of the establishment of a diffusion boundary layer which maintains the supply of one or more growth factors below a critical level². A diffusion boundary layer results because convective movement of liquids near a solid surface approaches zero. In these conditions, the rate of delivery of molecules to and from the surface is limited by the rate of diffusion through the boundary layer. Increased fluid velocity on confluent cells causes initiation of DNA synthesis^2,3, a finding apparently supporting the diffusion barrier hypothesis. Theoretically, increasing the fluid velocity over the cell layer should decrease the thickness of the hypothetical boundary layer and therefore increase the rate of delivery of a diffusion-limited growth-controlling substance⁴. However, in these experiments it was not possible to rule out an effect of fluid velocity on cell–cell contact. Yet if growth is a diffusion-limited process, then increasing viscosity of the cell culture medium should decrease the saturation density. We report here that increased viscosity has no effect on saturation density or on the rate of cell growth, providing strong evidence against the diffusion barrier hypothesis.