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T Saga, D Scheurer, R Adler; Development and maintenance of outer segments by isolated chick embryo photoreceptor cells in culture.. Invest. Ophthalmol. Vis. Sci. 1996;37(4):561-573.
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PURPOSE: To investigate the capacity of isolated chick embryo photoreceptors to develop and maintain outer-segment processes in dissociated cell cultures, in the absence of pigment epithelial and glial cells. METHODS: Cells were obtained from the retinas of embryonic day (ED) 17 chick embryos, after the onset of outer-segment formation in vivo. After 5 to 12-minute incubation in Ca++ and Mg++-free Hank's balanced salt solution, neural retinas were freed from other optical tissues, including the pigment epithelium. Retinal cell suspensions were prepared by repeated pipetting after mild trypsinization and were grown in serum-containing medium on a polyornithine-coated substratum. Cell differentiation was evaluated using phase-contrast and transmission electron microscopes and by autoradiographic analysis of the uptake of putative amino acid neurotransmitters, lectin cytochemical analysis, and immunocytochemical analysis with rod and cone-specific antibodies. Cells isolated from ED 8 retinas, before the onset of outer-segment formation in vivo, were also studied. RESULTS: At culture onset, ED 17 cells appeared morphologically undifferentiated and devoid of processes; differentiated features could be detected after 24 to 48 hours in vitro. Photoreceptor cells were the most abundant cell type after 6 days in vitro, followed by nonphotoreceptor multipolar neurons and morphologically undifferentiated cells. Autoradiographic analysis showed extensive Na+ -dependent uptake of (2,3,4-(3)H)gamma- aminobutyric acid in nonphotoreceptor neurons, whereas photoreceptors were labeled predominantly with 3H-glutamate. Most of the photoreceptors were labeled with fluorescent peanut lectin and with a sheep polyclonal antibody against bovine rhodopsin. Subsets of photoreceptors, on the other hand, were immunoreactive with cone- or rod-specific monoclonal antibodies COS-1, OS-2, 50-1B11, or Rho-4D2. Approximately 50% to 65% of the photoreceptors positive with these monoclonal antibodies showed a remarkable polarization of immunoreactive materials, which accumulated predominantly, or even exclusively, in an outer-segment-like apical process. When viewed on the transmission electron microscope, these outer-segment-like processes appeared as distal expansions of the photoreceptor cilium and contained disc-like membranous profiles. Outer-segment-like processes also could be detected using the electron microscope and by immunocytochemical analysis of cultures of ED 8 retinal cells. CONCLUSIONS: After undergoing morphologic dedifferentiation as a result of tissue dissociation, isolated retinal photoreceptors, grown in the absence of contact-mediated cell interactions and of pigment epithelial and glial cells, can regenerate and maintain a highly polarized pattern of structural and molecular organization, including the formation of outer-segment-like processes. The cultures provide an experimental system for the investigation of cellular and molecular mechanisms regulating further development and maturation of these photoreceptor structures.
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