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Originally published In Press as doi:10.1167/iovs.08-2634 on April 22, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:3680-3687.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2634
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Enhancement on Primate Corneal Endothelial Cell Survival In Vitro by a ROCK Inhibitor

Naoki Okumura,1 Morio Ueno,1,2 Noriko Koizumi,3 Yuji Sakamoto,4 Kana Hirata,3 Junji Hamuro,1 and Shigeru Kinoshita1

1From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; the 2Department of Ophthalmology, National Center for Geriatrics and Gerontology, Obu, Japan; the 3Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan; and the 4Research Laboratory, Senju Pharmaceutical Co., Ltd., Kobe, Japan.

PURPOSE. The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs.

METHODS. MCECs of cynomolgus monkeys were cultured in a medium containing 10 µM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry.

RESULTS. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours.

CONCLUSIONS. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.






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