Shaking 3T3 cells: Further studies on diffusion boundary effects

Abstract

Confluent quiescent cultures of 3T3 cells can be stimulated to grow—with or without a medium change—simply by shaking (20 mm excursions, 3 per sec). Shaking without medium change, or with low serum concentration in fresh medium (4%), has the greatest effect on thymidine labeling and mitotic index compared with unshaken cultures, but high serum concentration (20%) stimulates both shaken and unshaken cultures equally. Even without medium change, the majority of cells can be stimulated to incorporate thymidine and to divide over 3 days, though initial shaking for 7 hr is sufficient to initiate growth. We conclude that growth in confluent 3T3 cell cultures is limited by a diffusion barrier in the microenvironment, probably affecting uptake of a serum component. The limitation can be overcome by increased fluid velocity as an alternative to raising the serum concentration. Shaking also causes increased cell movement and a change in cell morphology (which may be partly due to alterations in serum caused by shaking). Despite increased movement in the cell sheet, emigration from the border of a wound is diminished by shaking, suggesting that chemotaxis may be involved. To test whether loss of contact between the migrating cells is essential for growth stimulation at the border of a confluent layer, wounds were made in unshaken cultures in the presence of cytochalasin B, which prevents locomotion of cells and maintains the original topography. Nevertheless, the thymidine labeling index was still increased along the wound boundary, and included cells which were near but not on the edge. This supports the view that short range effects on cell growth at discontinuities in cell populations can be due to modification of a diffusion barrier and not simply alteration in cell contacts.