A method for the isolation of retinal pigment epithelial cells from adult rats.
OBJECTIVE
Isolating retinal pigment epithelium (RPE) cells from adult rat was the goal of this study.


METHODS
A modification of the procedure of Mayerson et al was the method employed. After the cornea and lens were removed, the eyecup was treated with hyaluronidase plus collagenase. The neural retina was carefully removed with little RPE attached. The eyecup, free of retina, was treated with the same enzyme cocktail that loosens any attached rod outer segments as well as the attachment of the RPE to Bruch's membrane. RPE cells were isolated in calcium-free medium as sheets of cells.


RESULTS
Light and electron microscopy revealed good cell morphology. Purity of the RPE preparation was established by microscopy, polyacrylamide gel electrophoresis, and lipid analysis. The viability of the isolated RPE was about 74% by trypan blue exclusion test.


CONCLUSIONS
This technique enables RPE to be isolated from adult rat in quantities sufficient for biochemical analysis.
Wang
N
N
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Koutz
C A
CA
Anderson
R E
RE
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure; D051381:Rats; D012374:Rod Cell Outer Segment / Q000737:chemistry
D005136:Eye Proteins; D010743:Phospholipids