Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.
OBJECTIVE
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 microM 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.
Okumura
Naoki
N
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Ueno
Morio
M
Koizumi
Noriko
N
Sakamoto
Yuji
Y
Hirata
Kana
K
Hamuro
Junji
J
Kinoshita
Shigeru
S
D000577:Amides / Q000494:pharmacology*; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002448:Cell Adhesion; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D019394:Ki-67 Antigen / Q000378:metabolism; D008252:Macaca fascicularis; D011725:Pyridines / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
D000577:Amides; D017304:Annexin A5; D004791:Enzyme Inhibitors; D019394:Ki-67 Antigen; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases