title: The effects of ROCK inhibitor Y-27632 on injectable spheroids of bovine corneal endothelial cells.
abstract: The spheroids of 3-dimensional culture and Rho-associated kinase (ROCK) inhibitor Y-27632 have shown many advantages for the promotion of cellular viability and proliferation. The objective of this study was to investigate the effect of Y-27632 on the growth and injectability of bovine corneal endothelial cells (B-CECs) maintained in vitro as spheroid cultures. Immunofluorescence staining showed that Y-27632 did not alter the cell type specificity of B-CECs, but it significantly enhanced B-CEC spherical viability and proliferation by a Live/Dead assay, 5-ethynyl-2'-deoxyuridine (EdU) labeling assay, and Cell Counting Kit-8 (CCK-8) assay. The uniform B-CEC spheroids could easily form in multiwall agarose micromolds and had a higher stemness potential than single B-CECs. Injectable B-CEC spheroids were able to form monolayer growth, and polygonal B-CECs completely covered culture plates or Descemet's membrane of decellularized corneas under inverted microscopy and scanning electron microscopy (SEM). B-CEC spheroids were generated from agarose microwells on day 1 and then adherent culture with Y-27632 for day 5. However, small B-CEC spheroids still existed on culture plates or decellularized corneas when B-CEC spheroids were cultured in the same condition except for absence of Y-27632. Our findings that CEC spheroids with Y-27632 are injectable in vitro have important implications for the favorable treatment of CEC deficiency.
authors.affiliation: 1 Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University , Guangzhou, 510632, China .
authors.forename: Yonglong
authors.initials: Y
authors.lastname: Guo
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Guo
authors.forename: Ruiling
authors.initials: R
authors.lastname: Lian
authors.forename: Shanyi
authors.initials: S
authors.lastname: Li
authors.forename: Chan
authors.initials: C
authors.lastname: Wang
authors.forename: Shiqi
authors.initials: S
authors.lastname: Zhang
authors.forename: Jiansu
authors.initials: J
authors.lastname: Chen
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000648:ultrastructure*; D004728:Endothelium, Corneal / Q000166:cytology; D011725:Pyridines / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000187:drug effects*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D000577:Amides; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases