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EM Barnett, X Luo, A Brasher, D Hicks, C Romano; Culture and Immunocytochemical Characterization of Retinal Neurons from Adult Human Eyes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):725.
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Purpose: Traditionally, retina neuronal cultures have been made with cells from fetal or embyonic animals. Cells from immature animals, however, may display different pharmacologic responses as compared with mature cells. The culture of neurons from human eyes is particularly advantageous in that it provides a more logical extrapolation of the data to human tissue and disease. Our goal is the culture and immunocytochemical identification of retinal neurons from adult human eyes as well as optimization of culture conditions for retinal ganglion cells. Methods: Retinal neurons were cultured from retinas isolated from adult human globes. Posterior segments were obtained from adult eyes without a history of eye disease through a local eyebank within 8 hours of harvest. The protocol for culture was adapted from that used to culture adult pig retinal neurons as recently published (IOVS, 42:1096, 2001). After 7 days, immunocytochemistry was performed on cultured cells using antibodies to retinal cell markers (including neurofilament-68, protein gene product 9.5, protein kinase C, arrestin, rhodopsin, and calbindin D28) to identify neuronal sub-types. Cultured cells were also grown in several different types of media and retinal ganglion cells counted at 2 weeks to determine optimal conditions for retinal ganglion cell culture. Results: Immunocytochemistry at day 7 reveals intact neurons from all retinal sub-types including retinal ganglion cells, amacrine cells, horizontal cells, and bipolar cells. Many neurons, particularly retinal ganglion cells, were noted to have impressive processes developed by that time. Most, if not all, cells plated survived beyond 1 week. The choice of media was shown to be an important determinant in optimizing retinal ganglion cell culture. Conclusion: Mixed retinal neuronal cultures can be made from adult human retina which contain all neuronal sub-types. Neurons survive for approximately two weeks allowing for experimental manipulation, including studies of cell survival/apoptosis. Culture of adult human retinal neurons should enable studies pertinent to a number of human eye diseases including glaucoma.
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