Culture of adult rabbit retinal glial cells: Methods and cellular origin of explant outgrowth

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Abstract
Culture methods to propagate glial cells from the avascular adult rabbit neural retina are described. To determine the site(s) in the retina from which the cells originated, retina fragments were retrieved from culture at intervals after explan-tation and processed for light microscopy to localize surviving cells. Tritiated-thymidine radioautography was used to determine the time of onset and the localization of proliferating cells in the isolated retina and in the early culture outgrowth. Although some glial cells located just subjacent to the inner limiting membrane and at the interface between the ganglion cell and inner plexiform layers were activated to DNA synthesis in three to five days after ex-plantation of the retina, the dominant cell type in the cultures derived from the retina appeared to be the Muller cell. Regionally, in isolated retina fragments, Muller cells became hyperplastic and formed tissue masses of proliferating cells surrounding photoreceptor remnants. These proliferating clusters could be retrieved from the culture medium after about 10 days in vitro and replated whereupon they attached to the culture substrate and gave rise to cellular outgrowths. The cells in the early explant outgrowth were heteromorphic, but passaged cultures contained a relatively homogeneous population of cells that exhibited a low maximal growth rate and senesced quickly in vitro.