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A. Zayas-Santiago, J. J. Kang Derwent; New Techniques for Maintaining Intact Adult Photoreceptors in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1266. doi: https://doi.org/.
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Intact adult photoreceptors in cell 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/or loss of outer segments during the cell culture process. The goal of this study is to develop a novel methodology to preserve photoreceptor cell’s native elongated shape in cell culture.
Photoreceptor cells were isolated from retinas of 21-days old Long Evans rats following a gentle dissociation process and seeded onto poly(dimethylsiloxane) (PDMS) microchanneled surfaces for 4 days. Parallel microchannels (1.25µm wide, 2µm deep, separated by 6µm wide ridges) were previously modified with selective deposition of wheat germ agglutinin (WGA) on the grooves, for selective attachment of rod cells. Attachment of cells to control flat PDMS WGA-modified surfaces was used as a control. The extent of the photoreceptor’s native shape preservation was evaluated through immunocytochemistry by expression of rod-specific-marker rhodopsin and 4',6-diamidino-2-phenyindole (DAPI).
A gentle retina dissociation consisting of low enzyme doses (0.1mg/mL Papain) and minimal mechanical trituration yielded an abundant population of photoreceptors showing intact structure (outer segments attached to cell bodies). After 4 days in culture, cells on control surfaces still maintained these characteristics; however the outer segments shrank in size. Cells on microchannels showed higher preference towards WGA-deposited grooves than to ridges. This prevented outer segment deformation and promoted the maintenance of the cell’s elongated shape.
Microchanneled surfaces facilitated rods to retain their in vivo shape and/or elongation. This suggests that an integrated approach of gentle dissociation and microfabrication techniques are promising in the quest of obtaining intact adult photoreceptors in vitro for possible application in cell replacement therapy.
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