Isolation and characterization of corneal endothelial cells from wild type and thrombospondin-1 deficient mice.
OBJECTIVE
To isolate and characterize primary corneal endothelial cells (CEC) from wild type and transgenic mice to facilitate the study of their properties in vitro.


METHODS
CEC were isolated from wild type or transgenic-immortomice corneas. The Descemet's membrane was gently peeled from the periphery of the cornea towards the central region and placed into wells of a 96 well tissue culture plate coated with fibronectin in growth medium. Cells that grew out were trypsinized and expanded on fibronectin-coated wells and used for further characterization. CEC were evaluated for expression and localization of specific markers and adhesion molecules by FACS analysis and indirect immunofluorescence staining. The migration properties of CEC were evaluated using a scratch wound and transwell assay, while their ability to undergo capillary morphogenesis was assessed on Matrigel.


RESULTS
Isolation of CEC from transgenic mice has been somewhat challenging and not previously reported. Here we describe a method for isolation of CEC from wild type and thrombospondin-1 deficient (TSP1-/-) immortomice. Our results indicate that nearly 100% of selected cells express B4-lectin and VE-cadherin, but not PECAM-1. These cells were successfully passaged and maintained in culture for several months without a significant loss in expression of these markers. The wild type CEC, like vascular EC, organized and formed a capillary-like cell network on Matrigel. The ability of the CEC from TSP1-/- mice to form such a network was somewhat compromised. This may be attributed, at least in part, to altered adhesive and migratory properties of these cells.


CONCLUSIONS
The CEC can be readily obtained from wild type and transgenic mice, which facilitate the comparison and identification of the physiologic role of specific genes in CEC function.
Scheef
Elizabeth A
EA
Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Huang
Qiong
Q
Wang
Shoujian
S
Sorenson
Christine M
CM
Sheibani
Nader
N
D000818:Animals; D015153:Blotting, Western; D002196:Capillaries / Q000502:physiology; D002465:Cell Movement; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen; D004338:Drug Combinations; D004728:Endothelium, Corneal / Q000098:blood supply / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D005109:Extracellular Matrix; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D007797:Laminin; D051379:Mice; D018345:Mice, Knockout; D018919:Neovascularization, Physiologic; D010641:Phenotype; D011509:Proteoglycans; D019700:Thrombospondin 1 / Q000172:deficiency*
D004338:Drug Combinations; D016326:Extracellular Matrix Proteins; D007797:Laminin; D011509:Proteoglycans; D019700:Thrombospondin 1; C059485:matrigel; D003094:Collagen