The growth and behaviour of rat retinal Müller cells in vitro 1. An improved method for isolation and culture

Eyeballs were enucleated from young (postnatal day 8–12) pigmented rats and the retinas were dissected free after soaking the globes overnight in growth medium. The retinas were digested with enzymes, dissociated and maintained in stationary culture in 10% serum supplemented growth medium. Cultures displayed extensive cellular outgrowth after 1–5 days, with abundant fusiform and epithelioid cells. Removal of aggregates and cellular debris after 6–7 days yielded a purified flat cell preparation, which could be maintained either as a primary culture for several weeks or passaged repeatedly as rapidly proliferating epithelioid cells. Staining with monoclonal antibodies RET-G1, G3 and G7, and polyclonal S-100, glutamine synthetase and carbonic acid anhydrase antisera, all markers for Müller cells, showed positive labelling of all cells present in these purified cultures, both primary and passaged cells. This contrasted with the use of RET-G2, anti Factor VIII and anti glial fibrillary acidic protein (GFAP) antibodies. RET-G2, another Müller cell marker, failed to recognize passaged cells. Anti Factor VIII also did not label any cells, and anti GFAP stained very few cells: these remained associated with aggregated material so that vigorous washing to remove loosely adherent tissue from primary cultures resulted in the total absence of GFAP positive cells. In addition, no GFAP positive cells were detected in passaged cells or in cells regrown following freezing and storage. The Müller cell nature of these flat cells in soaked retinal cultures was further supported by the specific uptake of 5-bromo-deoxyuridine by nuclei located within the inner nuclear layer of retinal fragments in vitro. Hence the soaking treatment greatly reduces the number of surviving astrocytes whilst stimulating the rapid growth of cells expressing many properties of mature retinal Müller cells.