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Wing Yan Yu, Carl M. Sheridan, Ian Grierson, Amy C. Lo, David Wong; Enhanced Survival and Expansion of Bovine Corneal Endothelial Progenitors using Accutase. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6019.
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© ARVO (1962-2015); The Authors (2016-present)
Progenitors from the bovine corneal endothelium (CE) have been successfully isolated and expanded in suspension culture as spheres. Trypsin was commonly used to passage the spheres. However, Accutase, which contains proteases and collagenases, was reported to be less damaging to cells. In this study, the progenitor viability and expansion rate were compared using the two enzymatic dissociation approaches.
Primary peripheral CE cells were isolated from fresh bovine eyes. Cells of passage 1-3 were harvested either with trypsin or Accutase for sphere culture in serum-free media supplemented with B27, epidermal growth factor and fibroblast growth factor-2. After 7 days, the floating spheres were collected, dissociated into single cells with trypsin or Accutase, and sub-cultured. The number of spheres and viable cells were counted for each passage. Cell viability after the enzymatic treatments was determined by trypan blue exclusion test. The results were obtained from three individual experiments conducted in triplicate. Stem cell marker expression in cells that migrated from the spheres was investigated by immunocytochemistry.
The percentage of surviving cell was higher after Accutase (90-95%) than trypsin (75-80%) digestion. With the same initial seeding density at 10cells/µl, significantly higher number of spheres was formed in Accutase-treated primary (p<0.01), secondary (p<0.05) and tertiary (p<0.05) cultures than trypsinized cultures. An increased expansion rate of 9-fold was observed in primary spheres dissociated by Accutase compared to 6-fold by trypsin after 7 days. In passage 4, spheres with smaller diameter and less round in shape were seen and a large proportion of the cells died, both with Accutase or trypsin. Nestin, telomerase and beta-III tubulin expression were detected in the cells that migrated from the spheres. There was no obvious difference between the stem cell marker expression of cells in spheres treated with Accutase and trypsin.
Accutase significantly enhanced the bovine CE progenitor survival and expansion and thus is a better dissociation reagent for the stem cell expansion than trypsin. The limited number of passages may suggest that the progenitors being isolated had relatively limited self-renewal capacity or a more optimal culturing condition needs to be further elucidated.
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