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M Valtink, MO Karl, J Bednarz, K Engelmann; Short-Term Influence of Culture Media and Supplements on Phagocytosis of Photoreceptor Outer Segments by Cultured Human Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):716.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:It is imperative that cell culture conditons allow proliferation and maintenance of specific cell functions without dedifferentation, if transplantation of cultured retinal pigment epithelium (RPE) is to be successful in the treatment of retinal diseases. In this study we investigated the ability of complex and defined culture media to support phagocytosis. A method was established to measure binding and internalisation of POS simultaneously. Methods:Photoreceptor outer segments (POS) were isolated from porcine retinae and labelled with SNAFL®-2 (MoBiTec). SV40-transfected human RPE cells were seeded at a density of 10,000 cells / well on gelatin-coated 96 well-plates and cultured with medium F99+10% FCS. At subconfluence, serum was omitted for 12 h, followed by an incubation period of 12 h with the test media. Then POS (5x106 /100 µl and well) were added for 4 h and phagocytosis was measured by means of a cytofluorometer (Cytofluor®4000, PerSeptive Biosystems). Test media were F99 and Endothelial-SFM (Invitrogen), a commercially available medium designed for serum-free cell culture. When indicated, these media were further supplemented with fetal calf serum (FCS), insulin and pyruvate, retinal extract (RE) or choroid-conditioned medium (CCM). Results:Phagocytosis of RPE cultured in medium F99 was increased more than 3-fold after supplementation with up to 10% FCS. Further supplementation with insulin and pyruvate led to only a 2-fold increase compared to non-supplemented cultures. Quenching of the serum-induced stimulation could further be increased by addition of CCM, whereas adding RE instead of CCM led to a recovery in phagocytosis to 3-fold that of serum-free F99. In comparison to serum-free F99, endothelial-SFM showed a slightly higher stimulation of phagocytosis (1.5-fold). A serum stimulation was less pronounced using medium Endothelial-SFM in contrast to F99. Conclusion:In previous studies we could show that stimulation of proliferation by media and supplements is as follows F99 < SFM < media plus serum < media plus serum plus insulin and pyruvate < plus further addition of conditioned media. Using the described assay we demonstrate an inverse effect of proliferation stimulating media or supplements on internalisation of POS. In contrast, serum has been shown to stimulate both, proliferation and phagocytosis. Further studies on interaction of media, supplements and serum on phagocytosis based on these observations are needed. The established method allows investigating the effects of various supplements at a time without much effort.
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