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Y. Luo, M. Fukuhara, Y. Zhou, C. Rahner, L. Rizzolo; Growth Conditions Affect the Formation of Tight Junctions in ARPE–19 Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3034.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ARPE–19 cells obtained from the American Type Culture Collection are undifferentiated when grown under the standard culture conditions reported for these cells. We examined whether improved culture conditions could promote redifferentiation and provide a model to study the assembly of tight junctions. Methods: ARPE–19 was grown on laminin–coated Transwell filters in DMEM/F12 supplemented with 2% fetal bovine serum (FBS). On the forth day post–plating three different culture media were used to maintain the cultures: DMEM/F12 with 1% FBS, a serum free medium, SF3 medium (Peng et. al, IOVS 44:808–17, 2003), special medium (serum–free medium formulated and generously supply by J. Tombran–Tink, Univ Missouri). Culture media were changed weekly. After 3 and 6 weeks of culture, immunoblot and immunocytochemistry were used to analyze the expression of actin JAM 1, PAR–3, AF–6 and ZO–1. Effects on tight junction function were analyzed by measuring the transepithelial electrical resistance and permeability of horseradish peroxidase. Results: Three weeks after culture, ARPE–19 cells under 1% FBS DMEM/F12 displayed irregular cell morphology with numerous actin stress fibers. Cells under SF3 and special medium displayed more cortical actin and a more uniform, cobblestone appearance. Six weeks after culture, some cells under SF3 medium displayed prominent apical microvilli and the most pigmentation among three groups. The expression of JAM–1, PAR–3, AF–6 and ZO–1 proteins increased slightly with the increased culture time. ARPE–19 cells maintained with special medium had the highest expression of these tight junction protein, and JAM–1, PAR–3, AF–6, ZO–1 proteins were more specifically colocalized and distributed on lateral membranes. TER of ARPE–19 monolayers maintained in special medium had the highest TER and lowest permeability to horseradish peroxidase. Conclusions: Despite the expression tight junctional proteins, especially those thought to regulated junction assembly, cells in FBS had a non–epitheliod morphology and no barrier properties. Serum starvation and specialized media induced the expression of some factor(s) that was required in addition. Therefore, this model has the potential to reveal mechanisms of junction assembly.
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