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G. T. Grottone, P. C. Cristovam, M. F. Forni, M. C. Sogayar, J. A. P. Gomes; Isolation and Migration Pattern of Cultured Human Endothelial Corneal Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1822.
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Test and validate a new enzymatic method to isolate and seed human corneal endothelial cells. Verify migration and morphological changes during cell culture. Characterize cultured cells examining differences between central area and peripheral areas.
Ten corneas of five different patients were used for this experiment. Descemet's membrane and endothelial cells were isolated on dissecting microscope. After peeling, these layers were seeded separately at 6-well plates filled with Collagenase A for overnight digestion. The solution was centrifuged for 6 minutes at 2400 rpm. The cells from 6 corneas were plated in 24-well Matrigel coated plates. Cells from other 2 corneas were plated in 24-well uncoated plates. After the first 48 hours, media was replaced every other day. Migration studies were followed by phase contrast microscopy and staining with Rhodamine B 4%. The last two corneas were used for immunohistochemistry with vimentin and b3-tubulin.
Cells were seeded successfully in 24-well plates with Matrigel and cells adhered after 24 hours. Cells seeded under uncoated plates were not successful in adhering the plates and died after the first week in suspension. Cells spread after one week in culture in a centripetal orientation. The cell density was variable, showing a higher density at the center of the colony and a lower density at the periphery. After two weeks in culture, most samples started to change the morphology at the periphery of colonies in a low cell density. These cells became elongated resembling a cobblestone-like or fibroblastic shape. Staining with rhodamine-B enhanced the visualization of colony morphology and their characteristics. Immunohistochemistry was positive for vimentin and b3-tubulin in studied cultures.
Usage of collagenase A enzymatic method is reliable and reproducible. ECM coating with Matrigel aided the adherence of isolated cells. Cells spread outward of the initial adherence site until they reached confluence with another colony. Immunohistochemistry validated the cultured cells as human endothelial corneal cells, staining positive to neural crest and mesenchymal markers.
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