Cell culture conditions affect RPE phagocytic function.

Background: Changes in the phenotype of retinal pigment epithelium (RPE) cells in vitro are associated with medium conditions and changes in function. Main goals in RPE tissue engineering are cell propagation in serum-free defined culture conditions, resulting in cells exhibiting differentiated morphology and functioning in vitro.

Methods: To compare the effects of various media and supplements on cell function, an optimized high-throughput phagocytosis assay was developed. Adult human SV40-RPE cells were cultured. Test media included: MEM(E), DMEM, F99, SFM and hSFM, with or without supplements. SNAFL-2 labelled OS were added to RPE in vitro for 4 h and phagocytic binding and uptake were measured.

Results: RPE phagocytosis was of different magnitude depending on the serum-free basic cell culture media in the following order: hSFM, SFM > DMEM, MEM > F99. Choroid-conditioned medium (ChCM) decreased phagocytosis dose dependently. Whereas 1% retinal extract (RE) supplementation increased, higher concentrations decreased phagocytosis. Addition of 10% FCS increased phagocytosis. 15% ChCM quenched the stimulation induced by 10% FCS, an effect which could be reversed by the addition of 1% RE.

Conclusions: Cell culture media and RPE environmental factors exert substantial and differential alteration of RPE phagocytic ability. Phagocytosis in a serum-free defined medium is superior to unsupplemented basic media, but still differs from serum-supplemented media (F99RPE) designed for cell propagation. We conclude that media SFM or hSFM promoted phagocytosis most, and application of FCS or 1% RE supports phagocytosis. Unknown factors from neighbouring tissues (retina and choroid) affect phagocytosis differently, suggesting a role in retinal pathogenesis. The results will support identification of specific environmental factors and facilitate design of cell culture media.