A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers

PURPOSE: The retinal pigment epithelium (RPE) in vivo is known to have pola rized membrane domains that are essential for its normal function. Unless t he proper cell culture conditions are used, these polarized features are of ten lost. In the past, the use of Chee's Essential Medium (CEM) in our RPE cultures has produced functional polarity of the cell monolayers. Unfortuna tely, except by custom formulation, which is costly, this product is no lon ger commercially available. We therefore sought to develop a replacement cu lture medium that would support morphological and functional polarity of RP E membrane domains when the cells are removed from the in vivo milieu. METHODS: To test the performance of this CEM replacement medium in comparis on with three other culture media, we grew fetal human RPE to confluence on Millipore MillicellTM culture wells. We then used Na,K ATPase as a membran e domain marker by displaying it with polyclonal antibodies. This marker wa s chosen because it is not always properly polarized in culture. Immunofluo rescence was imaged by laser confocal microscopy of whole mounted intact mo nolayers on their Millicell supports. We also used transepithelial resistan ce (TER) as a measurement of functional polarity as well as bestrophin prot ein expression as an index of cell differentiation. The expression of Na,K ATPase and bestrophin was confirmed by Western blot analysis of whole RPE c ell extracts. RESULTS: Immunofluorescence labeling of cultured RPE Na,K ATPase was observ ed exclusively on the apical membrane when the CEM replacement or DMEM with high glucose was used. However Na,K ATPase was not completely polarized in DMEM/F12 medium and the cells did not express detectable Na,K ATPase in DM EM with low glucose. Western blots showed that Na,K ATPase was expressed at similar levels in CEM replacement, DMEM with high glucose and DMEM/F12 as indicated by the intensity of an approximately 100 kDa band representing th e a subunit. The CEM replacement gave superior TERs as well, ranging from a bout 2 to 5.6 fold higher than the other media. Bestrophin protein was read ily detectable by Western blot in CEM replacement medium whereas it was bar ely detectable in DMEM/F12 and undetectable in DMEM with high and low gluco se. CONCLUSIONS: We have provided immunocytochemical evidence that the CEM repl acement medium supports the appropriate membrane domain expression of Na,K ATPase when the cells are grown on Millicell chambers. Excellent TERs and r obust expression of bestrophin are also observed. This combination of featu res was not observed when other, standard culture media were used. The resu lts suggest that, under these conditions, cultured human RPE develops a hig hly differentiated and functional polarity appropriate for the in vitro mod eling of RPE in vivo function.