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V Doubilei, J Bednarz, M Valtink, V Zubaty, M Karl, K Engelmann, H Schaefer; Serum-Free Cultivation of Adult Human Retinal Pigment Epithelial (RPE) Cells for Transplantation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3450.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Transplantation of RPE cells as a possible therapeutic approach in cases of retinal dystrophies requires a high number of differentiated, functioning RPE cells. We present a method for isolation and cultivation of adult human RPE cells in a serum-free medium. This method supports maintenance or restoration of differentiation and pigmentation of the cells in vitro. Methods: Human adult RPE cells were isolated from donor eyes by means of collagenase I and IV and seeded on fibronectin-coated culture plates in medium Human Endothelial-SFM (Human-SFM; Invitrogen), initially supplemented with 20 ng/ml bFGF and 10 ng/ml EGF. The cells were further cultured in Human-SFM without any supplementation. For subculture, cells were trypsinized and cultured serum-free as before. The morphology of cultured cells was monitored by light and electron microscopy (EM) and by immunohistochemical staining for cytokeratins 8, 18 and 19, HMB-45, and ZO-1 (tight junction (TJ) protein). RPE cells cultured in the previously described medium F99RPE served as a control. Early passage cultures were incubated with isolated porcine photoreceptor outer segments (POS) in order to determine phagocytotic ability. Results: Confluent cell cultures in Human-SFM displayed typical epithelial, polygonal morphology with strong contact inhibition and underwent up to 5 cumulative population doublings. Numerous small colony-forming cells with phase-bright cell borders were observed in all cultures. Proliferating cells lost their pigment during growth and exhibited repigmentation approx. 6 weeks after they reached confluence. The de novo synthesized pigment was predominantly observed in colony-forming cells. Repigmented cells did not proliferate under the described conditions. Cells expressed cytokeratins 8, 18 and 19, ZO-1, and colony-forming cells were transiently positive for HMB-45 during phases of increasing repigmentation. EM revealed numerous microvilli, TJ, increasing number of pigment granules and ingested POS. Microvilli, TJ and repigmentation were not seen in the cells cultured in medium F99RPE Conclusion: Adult human RPE cells can be cultured and expanded under serum-free conditions. The cultured cells exhibit typical morphological and functional features similar to those of RPE cells in vivo. They form a polarized monolayer with TJ and microvilli, are able to phagocytose POS, and regain their pigmentation. The presented method allows obtaining sufficient quantities of highly differentiated RPE cells for further RPE transplantation studies.
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