The Effects of ROCK Inhibitor Y-27632 on Injectable Spheroids of Bovine Corneal Endothelial Cells

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.