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N. Okumura, M. Ueno, N. Koizumi, H. Hisako, S. Kinoshita; A ROCK Inhibitor Enhances Primate Corneal Endothelial Cells Culture in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5041. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The transplantation of cultivated corneal endothelial cells (CECs) has gained attention for the treatment of patients with corneal endothelial dysfunction. Techniques for growing human CECs have been reported, however, it is still difficult to culture human CECs efficiently while keeping high cellular densities and favorable morphology. This current study was conducted to demonstrate the usefulness of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cellular proliferation of cultivated monkey corneal endothelial cells (MCECs), which, as with human CECs, are considered to have less proliferative potential.
MCECs of cynomolgus monkeys were removed from the Descemet’s membrane by dispase treatment and cultured in a medium containing 10 µM Y-27632. Culture medium without Y-27632 was used for the control cultures. The numbers of viable cells were determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corp., Madison, Wisconson) after 24 hours of primary and passage culture, respectively. The cell density of MCECs was examined during 6 times of passage culture. Cellular proliferations of passaged MCECs were determined by Ki67 expression by immunocytochemistry and flow cytometry (FCM). Annexin V-positive apoptotic cells were analyzed by FCM. The actin cytoskeleton organization and cellular morphology were evaluated by phalloidin staining.
The numbers of viable cultivated MCECs were enhanced by Y-27632 treatment after 24 hours of culture (39.5±0.15% and 18.9±2.8% for primary culture and passage culture, respectively; p<0.05 and p<0.01). Y-27632-treated MCECs showed higher cell density throughout the 6 times of passage period. In Y-27632-treated cultures, Ki67-positive cells were increased at 24 and 48 hours and annexin V-positive apoptotic cells were decreased at 24 hours. Phalloidin staining revealed that Y-27632-treated cells had more potentiated actin stress fibers.
We have demonstrated that Y-27632 treatment enhanced the cellular proliferation and prevented apoptosis of MCECs, which resulted in maintaining higher cell density in passage cultures. The Y-27632 treatment was also effective to promote actin cytoskeleton organization which might be effective for cellular morphology, movement, and adhesion of MCECs during the culture period. We consider our results are applicable for human CECs culture to promote regenerative medicine of corneal endothelium.
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