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Ayaka Kusakabe, Naoki Okumura, Hiroatsu Hirano, Noriko Koizumi, Shigeru Kinoshita; Purification of high cell density cultured corneal endothelial cells by density-gradient centrifugation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1167.
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We have initiated clinical research of a cultured corneal endothelial cells (CECs) injection therapy for treating corneal endothelial dysfunction. Cell density (CD) of CECs tends to decrease during serial passages for expansion culture, though transplantation of high CD-CECs is thought to be beneficial for the clinical outcome. The purpose of this study was to investigate the feasibility of density-gradient centrifugation to remove low CD-CECs and to purify high CD-CECs for clinical application.
A primary culture of human CECs (HCECs) obtained from donor corneas was passaged 5-6 times and then used for the experiments. The cultured HCECs were suspended in Opti-MEM® Reduced Serum Medium (Life Technologies), and OptiPrep™Density Gradient Medium was then added. After centrifugation at 800 g for 15 minutes, HCECs were recovered from the pellet and supernatant, respectively. The diameters of the HCECs were evaluated by flow cytometry. HCECs recovered from both the pellet and supernatant were cultured for 4 weeks, and CD and cell area were evaluated by Image J software. Expression of function-related proteins such as ZO-1 and Na+/K+-ATPase was examined by immunocytochemistry.
Low CD-CECs were observed as a mosaic pattern in high CD-CECs after 3 passages. The mean diameter of pellet-HCECs was significantly smaller than that of supernatant-HCECs (42.9±0.17 μm and 49.5±0.31 μm,) (p<0.01). After 4-weeks culture, the mean CD of the pellet-HCECs and supernatant-HCECs was 1584.5±69.5 cells/mm2 and 827.8±394.1 cells/mm2 , respectively (p<0.01). The mean cell area of the pellet-HCECs (interquartile range) was lower than that of supernatant-HCECs (301.9 (244.6-355.2) μm2 and 635.0 (365.6-1354.6) μm2, respectively). Immunocytochemistry showed that pellet-HCECs express hexagonal morphology and ZO-1 and Na+/K+-ATPase on their plasma membranes, while hexagonal morphology and expression of ZO-1 and Na+/K+-ATPase were partially disrupted in supernatant-HCECs.
Our findings indicate that density-gradient centrifugation enables the purification of high-CD HCECs by removing low-CD HCECs. Though a protocol for clinical application needs to be optimized, density-gradient centrifugation is useful to purify high-CD HCECs for tissue engineering therapy for corneal endothelial dysfunction.
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