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

[摘要] Purpose: Intact adult photoreceptors inculture can be a valuable tool in the search of therapies for retinaldegenerations. The major challenge in this technique is thatphotoreceptors undergo an alteration in cytoarchitecture and loss ofouter segment during the cell culture process. This study comparedtechniques for the isolation of photoreceptor cells from adult ratretinas to determine which technique yields the highest percent ofstructurally well preserved cells in vitro. In addition, the role oflight exposure during the dissociation and culture process wasinvestigated to minimize photoreceptor cell deformation over time inculture. Methods: Photoreceptor cells from adultrat retinas were isolated and quantified using three dissociationtechniques: enzymatic dissociation with gentle pipeting; enzymaticdissociation with gentle pipeting and centrifugation; andnon-enzymatic dissociation with gentle pipeting. To evaluate theeffect of light exposure on cell deformation, we performeddissociations and cell seeding both in dark- and light-adaptedconditions and measured the deformation of photoreceptors over a 12 hperiod right after dissociation. Cell viability in both conditions wasevaluated after 4 and 7 days in culture. Preservation of cell structurein culture was assessed by immunofluorescence labeling of cells withanti-rhodopsin and 4',6-diamidino-2-phenylindole (DAPI) nuclearstaining. Results: An enzymatic technique followedby gentle pipeting or mechanical trituration yielded the highest numberof intact elongated photoreceptors right after dissociation. Datasuggested that centrifugation after the dissociation contributed tocell deformation immediately after isolation. Immunohistochemistryresults showed that cells had deformed into a circular shape by 2 daysafter seeding. However, photoreceptors isolated in dark conditionsmaintained their elongated shape, even 7 days after seeding. Performingexperiments in dark also promoted a higher number of cells to remainviable with time. Conclusions: The current studydemonstrated the importance of proper isolation techniques to obtainthe maximum amount of intact photoreceptor cells. The data suggestedthat a gentle dissociation technique, consisting of enzymatic treatmentfollowed by moderate pipeting of the retinal tissue, may be the key toobtain a high number of intact or structurally preservedphotoreceptors. Furthermore, isolation and cell culture proceduresperformed under dark conditions may facilitate to maintain high numberof elongated photoreceptor cells in vitro.

[学科分类] 生物化学/生物物理