Survival of the retinal pigment epithelium in vitro: comparison of freshly isolated and subcultured cells.

摘要: Cells of the retinal pigment epithelium (RPE) are generated prenatally and generally survive lifetime individual without undergoing proliferation or replacement. Therefore, mechanisms promoting RPE cell survival longevity in vivo may be distinct from, a limited subset of, known to promote proliferative cells culture. To identify specific factors that sustain viability independent effects on division, we studied low-density suspension culture, which is inhibited. Single from Xenopus laevis eyes were plated onto non-adhesive surface protein-free medium, then assayed for using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell these cultures was essentially undiminished over initial 2 days. However, by approximately 1 week only an average 53% remained alive. Plating fibronectin-coated substratum significantly enhanced survival, such number alive at 80-90% level. Essentially identical results obtained with laminin- collagen IV-coated substrata, insulin (5 microg ml(-1)) medium. The absence division confirmed counting BrdU incorporation experiments. Interestingly, derived monolayers previously established microporous membrane filters, lost much faster (average 80% dead 3 days), showed relatively greater response extracellular matrix proteins (five-fold increase days). Enhanced fibronectin required spreading substratum, rather than mere adherence, as there high correlation between percentage spread MTT-positive (r=0.940). apparently preventing initiation programmed death: unattached non-viable culture exhibited morphological features expected apoptosis, well positive staining TUNEL reaction. These studies demonstrate that, several shown maintain proliferating cultures, some have their effect, least part, cells. increased susceptibility subcultured death has implications clinical transplantation applications require manipulation vitro.