Survival and differentiation of purified retinal ganglion cells in a chemically defined microenvironment.

PURPOSE

To develop a fully defined medium and substratum that will support the survival and differentiation of purified retinal ganglion cells (RGCs) from newborn rats, and to evaluate beneficial effects of various hormones.

METHODS

RGCs were purified from papain-dissociated retinal cells by a two-stage panning method and cultured in a fully defined medium on a substratum precoated with polyornithine and laminin. RGC purity was assessed by prelabeling RGCs with retrogradely transported horseradish peroxidase and by anti-Thy-1 immunocytochemistry. Various hormones were evaluated for their ability to promote RGC survival by adding them to the culture medium.

RESULTS

Peroxidase labeling yielded purity estimates between 96% and 100%. Within 2 days, two distinct cell types are easily identified: a small cell characterized by thin, unbranched neurites and a large cell characterize by extensively branched neurites. Most small and large cells were round; however, some had elongated cell bodies. Both small and large cells express Thy-1 on their surfaces. Size-histogram analysis of all cells yielded a bimodal distribution. Survival was enhanced by seeding at higher densities and by the addition of hydrocortisone and progesterone to the cultures.

CONCLUSIONS

This defined culture system should facilitate further studies on the direct mechanisms regulating retinal ganglion cell survival and differentiation.