Induction of Differentiation by Pyruvate and DMEM in the Human Retinal Pigment Epithelium Cell Line ARPE-19

被引:82
作者
Ahmado, Ahmad [1 ,2 ]
Carr, Amanda-Jayne
Vugler, Anthony A.
Semo, Ma'ayan
Gias, Carlos
Lawrence, Jean M.
Chen, Li Li
Chen, Fred K. [2 ]
Turowski, Patric [3 ]
da Cruz, Lyndon [2 ]
Coffey, Peter J.
机构
[1] UCL, Inst Ophthalmol, Dept Ocular Biol & Therapeut, London Project Cure Blindness, London EC1V 9EL, England
[2] Moorfields Eye Hosp, Dept Vitreoretinal Surg, London, England
[3] UCL, Dept Cell Biol, London EC1V 9EL, England
关键词
ENDOTHELIAL GROWTH-FACTOR; OXIDATIVE STRESS; IN-VITRO; HYDROGEN-PEROXIDE; VISUAL CYCLE; RPE CELLS; RCS RAT; MACULAR DEGENERATION; CULTURE-CONDITIONS; FACTOR SECRETION;
D O I
10.1167/iovs.10-6374
中图分类号
R77 [眼科学];
学科分类号
100212 ;
摘要
PURPOSE. Cultured retinal pigment epithelium (RPE) may become a therapeutic option for transplantation in retinal disease. However maintaining a native RPE phenotype in vitro has proven challenging. The human RPE cell-line ARPE-19 is used widely as an alternative to primary RPE. It is grown in DMEM/F12 medium as standard, but its phenotype is dependent on culture conditions, and many differentiation markers are usually absent. The purpose of this study was to examine how this sensitive phenotype of ARPE-19 can be modulated by growth media with or without the metabolite pyruvate to elucidate better RPE growth conditions. METHODS. ARPE-19 cells at passages p22 to p28 were cultured on filters for up to 3 months in DMEM/F12 or DMEM media with or without pyruvate and 1% fetal calf serum. Assessment of differentiation was performed using pigmentation, immunocytochemistry, protein/mRNA expression, transepithelial resistance, VEGF secretion, and ultrastructure. RESULTS. Pyruvate, in combination with DMEM, induced dark pigmentation and promoted differentiation markers such as CRALBP and MerTK. Importantly, RPE65 protein was detected by Western blotting and was enhanced by pyruvate, high glucose, and DMEM. ARPE-19 cells maintained in this medium could also phagocytose human photoreceptor outer segments (POS). VEGF secretion was greater in DMEM cultures and was affected by glucose but not by pyruvate. Pigmentation never occurred in DMEM/F12. CONCLUSIONS. This study demonstrated important differentiation markers, including pigmentation and Western blots of RPE65 protein, and showed human POS phagocytosis in ARPE-19 cultures using a simple differentiation protocol. The results favor the use of high-glucose DMEM with pyruvate for future RPE differentiation studies. (Invest Ophthalmol Vis Sci. 2011;52:7148-7159) DOI:10.1167/iovs.10-6374
引用
收藏
页码:7148 / 7159
页数:12
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