Maintenance of retinoid metabolism in human retinal pigment epithelium cell culture

If transplantation of cultured retinal pigment epithelium (RPE) or iris pig ment epithelium (IPE) is to be successful in the treatment of ocular diseas e, it is imperative to demonstrate that these cells can perform all of thei r necessary metabolic functions. Unfortunately, a critical function of the RPE, retinoid metabolism, is often lost rapidly in culture. We have examine d whether or not nonspecific proteolytic enzymes commonly used in cell isol ation and serial passaging may be responsible for this loss of function, an d we have investigated novel isolation and passaging techniques which can a lleviate 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 o n inserts coated with a layer of thermally responsive poly(N-isopropylacryl amide-co-cinnamoylcarbamidemethylstyrene). Upon confluence, cells were deta ched either by trypsinization or by lowering dish temperature. Retinoid met abolism of cells was assessed after isolation and culture by incubating mem brane fractions with H-3-all-trans-retinol. Retinoid metabolism was also me asured 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 collagenas e/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 cul tured 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 i nto individual cells, but these cells showed poor retinoid metabolism, incl uding no detectable retinyl eaters or 11-cis-retinoid isomers. IPE, CE and Hepa 6 did not show any retinoid metabolism. D407 and HepG2 produced retina ls, but not the 11-cis isomer. RPE cells isolated using trypsin lose the ability to form critical intermed iates in the visual cycle. Collagenase/hyaluronidase or nonenzymatic cell i solation techniques enable these functions to be maintained. After cell cul ture, thermally responsive surfaces allow nonenzymatic cell detachment and excellent maintenance of retinoid metabolism. (C) 1999 Academic Press.