title: Maintenance of retinoid metabolism in human retinal pigment epithelium cell culture.
abstract: If transplantation of cultured retinal pigment epithelium (RPE) or iris pigment epithelium (IPE) is to be successful in the treatment of ocular disease, it is imperative to demonstrate that these cells can perform all of their necessary metabolic functions. Unfortunately, a critical function of the RPE, retinoid metabolism, is often lost rapidly in culture. We have examined whether or not nonspecific proteolytic enzymes commonly used in cell isolation and serial passaging may be responsible for this loss of function, and we have investigated novel isolation and passaging techniques which can alleviate this loss of retinoid metabolism.RPE cells were obtained from human donor eyes by enzymatic and nonenzymatic methods. Cells were cultured either on control tissue culture inserts or on inserts coated with a layer of thermally responsive poly(N -isopropylacrylamide-co-cinnamoylcarbamidemethylstyrene). Upon confluence, cells were detached either by trypsinization or by lowering dish temperature. Retinoid metabolism of cells was assessed after isolation and culture by incubating membrane fractions with3H-all- trans -retinol. Retinoid metabolism was also measured in freshly isolated IPE, corneal endothelium (CE), an RPE cell line (D407), and two hepatocyte cell lines (Hepa 6 and HepG2). Membrane fractions from cells isolated nonenzymatically or using collagenase/hyaluronidase formed 11- cis -retinol, retinal isomers and retinyl esters. Retinoid metabolism of RPE cells freshly isolated by trypsinization showed no 11- cis -retinal and little 11- cis -retinol formation. Nondamaged cells cultured on thermally responsive surfaces detached in sheets upon temperature change. They showed metabolism similar to that of cells freshly isolated by nonenzymatic means. After trypsinization, confluent cultures dissociated into individual cells, but these cells showed poor retinoid metabolism, including no detectable retinyl esters or 11- cis -retinoid isomers. IPE, CE and Hepa 6 did not show any retinoid metabolism. D407 and HepG2 produced retinals, but not the 11- cis isomer.RPE cells isolated using trypsin lose the ability to form critical intermediates in the visual cycle. Collagenase/hyaluronidase or nonenzymatic cell isolation techniques enable these functions to be maintained. After cell culture, thermally responsive surfaces allow nonenzymatic cell detachment and excellent maintenance of retinoid metabolism.
authors.affiliation: Center for Controlled Chemical Delivery, Department of Pharmaceutics, University of Utah, Salt Lake City, UT, 84112, USA.
authors.forename: H A
authors.initials: HA
authors.lastname: von Recum
authors.forename: T
authors.initials: T
authors.lastname: Okano
authors.forename: S W
authors.initials: SW
authors.lastname: Kim
authors.forename: P S
authors.initials: PS
authors.lastname: Bernstein
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D018929:Cell Culture Techniques; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D014801:Vitamin A / Q000378:metabolism
chemical_list: D012176:Retinoids; D014801:Vitamin A; D014357:Trypsin