SERUM-FREE MEDIA FOR CULTURING AND SERIAL-PASSAGING OF ADULT HUMAN RETINAL-PIGMENT EPITHELIUM

The ability of a chemically-defined serum-free culture medium to suppo rt the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied, Primary cultures o f adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellula r matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times b y trypsinization, and trypsin activity was quenched with aprotinin, Fi rst passage RPE cells were plated onto tissue-culture plastic precoate d with bovine corneal endothelial extracellular matrix or uncoated tis sue-culture plastic in 24 well plates at a density of 50 viable cells mm(-2). Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine seru m, For each medium plating, efficiencies were determined 24 hours afte r plating, and growth rates were determined on the first, third and se venth days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages, Seeding e fficiency on bovine corneal endothelial extracellular matrix-coated ti ssue-culture plastic and treated tissue-culture plastic were higher fo r chemically-defined serum-free culture medium (88.9 +/- 2.7% and 47.1 +/- 4.1%, respectively) and DMEM with serum (8.72 +/- 5-6 % and 52.9 +/- 10.5 %, respectively) than DMEM without serum (59.2 +/- 5.6 % and 33.1 +/- 6.9%, respectively; P < 0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM wi th serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7, Cells cultu red in DMEM without serum, eventually decreased in number. RPE maintai ned in chemically-defined serum-free culture medium maintained a consi stent proliferation rate, reached confluence, and retained an epithelo id morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached conf luence at 12+/-3 days on bovine corneal endothelial extracellular matr ix-coated tissue-culture plastic and 21+/-5 days on treated tissue-cul ture plastic, Confluent cultures were composed of small hexagonal cell s with epitheloid morphology on both substrates. We concluded that pri mary adult human RPE can be cultured in this chemically-defined serum- free culture medium. RPE will proliferate, reach confluence, retain th eir epitheloid morphology and can be serially passaged in the absence of serum. (C) 1998 Academic Press.