title: Phenotypes of trypsin- and collagenase-prepared bovine corneal endothelial cells in the presence of a selective Rho kinase inhibitor, Y-27632.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, National Taiwan University Hospital Hsin-Chu Branch, Hsin-Chu, Taiwan ; Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan ; Department of Ophthalmology, National Taiwan University Hospital College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chien-Chia
authors.initials: CC
authors.lastname: Su
authors.affiliation: Department of Ophthalmology, National Taiwan University Hospital College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chun-Wen
authors.initials: CW
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan.
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Ho
authors.affiliation: Department of Ophthalmology, National Taiwan University Hospital College of Medicine, National Taiwan University, Taipei, Taiwan ; National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Fung-Rong
authors.initials: FR
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology, Cathay General Hospital, Taipei, Taiwan.
authors.forename: Shwu-Huey
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Ophthalmology, National Taiwan University Hospital College of Medicine, National Taiwan University, Taipei, Taiwan ; National Taiwan University College of Medicine, Taipei, Taiwan ; Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan ; Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan.
authors.forename: I-Jong
authors.initials: IJ
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D017364:Collagenases; D003599:Cytoskeleton / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018994:Myosin Light Chains / Q000378:metabolism; D010641:Phenotype; D011725:Pyridines / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects; D014357:Trypsin; D062826:Zonula Occludens-1 Protein / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D000199:Actins; D000577:Amides; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D011725:Pyridines; D062826:Zonula Occludens-1 Protein; C108830:Y 27632; D054460:rho-Associated Kinases; D014357:Trypsin; D017364:Collagenases