Development of neonatal mouse retinal neurons and photoreceptors in low density cell culture

We describe here a culture method which allows the growth of dissociated mouse retinal neurons and photoreceptors in chemically defined medium. Neural retinas from 2-day-old C57/BL mice were dissected from other ocular tissues, including the pigment epithelium, and dissociated into a cell suspension after brief trypsination. Most cells attached as single, unaggregated units to substrata pretreated with polyornithine and the neurite-promoting factor (PNPF). The cells were cultured in serum-free, high pyruvate Dulbecco's modified Eagle's medium containing chemically defined supplements. Under these conditions, onset of cell process development was rapid, giving rise to extensive neurite networks. Three morphologically distinct cell types were apparent during the first week in vitro. Some cells retained a circular outline and failed to produce processes, while 50-60% of the cells developed as multipolar neurons showing a large cell body and several neurites. Approximately 90% of these cells reacted with an amacrine cell-specific monoclonal antibody. Some 30% of the cultured cells expressed phenotypic properties characteristic of rod photoreceptors, including a small cell body, an apical cilium, a short neurite with a spherule-like terminal body, and immunoreactivity with antibodies against opsin as well as a rod cell-specific monoclonal antibody. No further signs of outer segment differentiation were observed in these cells. Non-neuronal "flat" cells, which represented less than 0.5% of the total cell number, reacted with an antibody against the glial fibrillary acidic protein. The number of neurons and photoreceptors remained relatively stable during the first 4-7 days in vitro. During the second week in culture, however, there was specific degeneration of greater than 90% of the photoreceptor cells, while less than 20% of the multipolar neurons were similarly affected. Consequently, in addition to providing a system for studying the differentiation of retinal neurons and photoreceptors, the specific degeneration of photoreceptors in these mouse retinal cell cultures makes this system ideal for investigating factors influencing photoreceptor survival.