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Judith Zavala, Mariana Lopez-Martinez, Daniela Gomez-Elizondo, Maria Dolores Montalvo Parra, Jorge E Valdez; Collagen production and pump function of corneal endothelial cells cultured in a two-phase system. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1453.
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Corneal endothelial cell (CEC) isolation and harvest face the challenge of maintaining cell morphology, expression of characteristic molecular markers, and in vitro functionality. Here we investigated the production of collagen and Na+/K+-ATPase function in rabbit CEC cultured in a two-phase approach.
Corneal endothelium form 3 White New Zealand rabbit 3 month-old was isolated, enzymatically digested using trypsin and collagenase I, and cultured in OptiMEM-I, 8% fetal bovine serum, 1% antibiotics, 5 ng/ml EGF, 20 ng/ml NGF, 20 μg/ml ascorbic acid, and 200 μg/ml calcium chloride until confluence. Cells were subcultured (1:3) using OptiMEM-I, 8% fetal bovine serum and 1% antibiotics until confluence. The cell lysate was used to determine total protein concentration by bicinchoninic acid assay, and pro-collagen I alpha 1 by ELISA. Na+/K+-ATPase was measured with a colorimetric assay. Human dermal fibroblasts (HDF) were used as control.
CEC harvested by the two-phase approach exhibited polygonal shape. Total protein concentration was 1,580 ug/100,000 cells in CEC and 490 ug/100,000 cells in HDF, pro-collagen I concentration was 150 pg/100,000 cells in CEC and 9 pg/100,000 cells in HDF. Na+/K+-ATPase concentration was 1.8 x 10-6 U/104 in CEC and 4.5x10-7 U/104 in HDF. Total protein, pro-collagen I, and Na+/K+-ATPase concentration were significative higher in CEC (P < 0.05).
CEC cultured in the two-phase approach exhibits polygonal shape, produces pro-collagen I and Na+/K+-ATPase function. These results demonstrate their potential use in preclinical models.
This is a 2020 ARVO Annual Meeting abstract.
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