J D Lindsey1, R N Weinreb. 1. Department of Ophthalmology, Glaucoma Center and Research Laboratories, La Jolla, California 92093-0946.
Abstract
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.
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.
Authors: Kimberly A Toops; Cynthia Berlinicke; Donald J Zack; Robert W Nickells Journal: Invest Ophthalmol Vis Sci Date: 2012-04-24 Impact factor: 4.799