Preservation of intact adult rat photoreceptors in vitro: Study of dissociation techniques and the effect of light

PURPOSE: Intact adult photoreceptors in culture can be a valuable tool in the search of therapies for retinal degenerations. The major challenge in this technique is that photoreceptors undergo an alteration in cytoarchitecture and loss of outer segment during the cell culture process. This study compared techniques for the isolation of photoreceptor cells from adult rat retinas to determine which technique yields the highest percent of structurally well preserved cells in vitro. In addition, the role of light exposure during the dissociation and culture process was investigated to minimize photoreceptor cell deformation over time in culture. METHODS: Photoreceptor cells from adult rat retinas were isolated and quantified using three dissociation techniques: enzymatic dissociation with gentle pipeting; enzymatic dissociation with gentle pipeting and centrifugation; and non-enzymatic dissociation with gentle pipeting. To evaluate the effect of light exposure on cell deformation, we performed dissociations and cell seeding both in dark- and light-adapted conditions and measured the deformation of photoreceptors over a 12 h period right after dissociation. Cell viability in both conditions was evaluated after 4 and 7 days in culture. Preservation of cell structure in culture was assessed by immunofluorescence labeling of cells with anti-rhodopsin and 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. RESULTS: An enzymatic technique followed by gentle pipeting or mechanical trituration yielded the highest number of intact elongated photoreceptors right after dissociation. Data suggested that centrifugation after the dissociation contributed to cell deformation immediately after isolation. Immunohistochemistry results showed that cells had deformed into a circular shape by 2 days after seeding. However, photoreceptors isolated in dark conditions maintained their elongated shape, even 7 days after seeding. Performing experiments in dark also promoted a higher number of cells to remain viable with time. CONCLUSIONS: The current study demonstrated the importance of proper isolation techniques to obtain the maximum amount of intact photoreceptor cells. The data suggested that a gentle dissociation technique, consisting of enzymatic treatment followed by moderate pipeting of the retinal tissue, may be the key to obtain a high number of intact or structurally preserved photoreceptors. Furthermore, isolation and cell culture procedures performed under dark conditions may facilitate to maintain high number of elongated photoreceptor cells in vitro.