Cell association increases RPE outgrowth from primary explant

Purpose. Most studies of cell growth of the RPE employ cultures which have been previously passaged, but here we investigated freshly-explanted RPE cells, which may have been growth-quiescent for years, within the eye to determine whether co-culture affects initial outgrowth in primary culture. Methods. Bovine or human RPE were co-cultured in primary culture with several cell types to test the effects of homologous or heterologous cell association. For bovine RPE, cell number was measured over 14 days in cultures of RPE alone, or RPE in co-culture with irradiated living cells, with fixed-killed cells, with cells separated from the RPE by a semipermeable membrane, or in medium conditioned by the cell types used for coculture. For human RPE, isolates from all donors were randomized, over a 13-month period, to co-culture or to culture alone. The number of cultures attaining a cell number at one month that was sufficient for further propagation was compared. Results. Co-culture with irradiated living cells increased the growth of primary cultures of both bovine and human RPE. Living cells were required; fixed-killed cells were ineffective. The outgrowth-promoting activity was not tissue or species specific, and it appeared to require close cell association between the RPE and the co-culture cell population. Conditioned media were ineffective and rather were slightly growth inhibitory. Primary RPE cells showed an earlier expression of vimentin (a marker of G₀-G₁ transition), more rapid cell spreading, and a greater increase in cell number between 7 and 14 days after explant when grown in co-culture than when cultured alone. Conclusions. The results indicate that cell association between non-mitotic RPE cells and previously cultured cells of many types increases the outgrowth of the RPE by accelerating the early stages of growth activation in vitro. Co-culture methods offer a practical means for increasing the likelihood of producing cultures from small RPE isolates. Further, should cells involved in proliferative pathologies in situ associate with nonmitotic RPE within the monolayer, the latter cells may also be activated, leading to an augmentation of the pathology.