Phenotypes of trypsin- and collagenase-prepared bovine corneal endothelial cells in the presence of a selective rho kinase inhibitor, Y-27632
Journal
Molecular Vision
Journal Volume
21
Pages
633-643
Date Issued
2015
Author(s)
Abstract
To optimize isolation of viable bovine corneal endothelial cells (BCECs), we evaluated the effectiveness of various preparation protocols. This entailed comparing the effects of collagenase A and trypsin in the presence and absence of a Rho kinase inhibitor, Y-27632, on proliferation and tight junctional and cytoskeletal integrity during their expansion. 5-bromo-2'-deoxyuridine (BrdU) incorporation evaluated cell proliferation. Western blot analysis evaluated F-actin, zonule occludin, and ZO-1 associated nucleic acid binding protein (ZONAB) and RhoA expression. Rho A pulldown assay evaluated Rho A activity. In the trypsin (TrypLE)-prepared BCECs, BrdU incorporation decreased whereas nuclear ZONAB expression increased and became stable from day 3 to 7. In contrast, in the collagenase-A-prepared BCECs, we observed preserved ZO-1 integrity, invariant nuclear ZONAB expression, and dense cortical F-actin expression, and BrdU incorporation was invariant from days 1 to 7. Y-27632 did not increase BrdU incorporation and nuclear ZONAB expression in the TrypLE-prepared and the collagenase-A-prepared BCECs. Moreover, Y-27632 increased irregular cellular morphology and downregulated the expression of ZO-1 in the collagenase-A-prepared BCECs from days 1 to 7. Y-27632 inhibited RhoA activation irrespective of whether the cells were isolated with trypsin or collagenase A. It is preferable to isolate BCECs with collagenase A and expand them without Y-27632. With this protocol, proliferative activity and tight junctional and cytoskeletal integrity are better preserved than if trypsin is used in the presence or absence of Y-27632.
SDGs
Publisher
Molecular Vision
Type
journal article