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.
Karl
Mike O
MO
Department of Ophthalmology, Cornea Bank, University Eye Clinic Eppendorf, Martinistrasse 52, Hamburg 20246, Germany. mail@mikeology.de
Valtink
Monika
M
Bednarz
Jürgen
J
Engelmann
Katrin
K
D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D005260:Female; D006801:Humans; D010587:Phagocytosis / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013539:Simian virus 40; D013552:Swine; D014162:Transfection
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free