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Bikash R. Pattnaik, Fan Yang, Sara Tokarz, Simran Brar, Matti P. Asuma, Marjorie Smithhisler, De-Ann M. Pillers; Characterization of Commercially Available Human RPE Cell Culture from LONZA®. Invest. Ophthalmol. Vis. Sci. 2011;52(14):871.
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© ARVO (1962-2015); The Authors (2016-present)
Malfunction of retinal pigment epithelium (RPE) cells distorts the function of neurosensory retina and hence affects our vision. A viable approach to study disease mechanism and RPE physiology is to make available suitable RPE cells in culture which can also be used in gene and transplantation therapy. We have characterized the cryopreserved Clonetics® Human Retinal Pigment Epithelial (hRPE) cells (# 00194987) from LONZA Walkersville Inc.
Human RPE cells were isolated and cultured using optimized RtEGMTM growth medium BulletKit® (# 00195409). RPE cells were characterized by immunohistochemistry of pancytokeratin, Na+K+ATPase, ZO-1, CD31 and fibroblast marker for cell identity and purity. After the cells formed a tight monolayer with hexagonal morphology, they were enzymatically dispersed and used for electrophysiology analysis using standard whole cell patch-clamp method. RPE specific transcripts were detected by RT-PCR.
Human RPE cells showed pigmented hexagonal monolayer after 4-6 days in culture. These cells expressed Kir7.1, bestrophin, pancytokeratin, Na+K+ATPase and ZO-1 but not endothelial and fibroblast markers. Unlike other RPE cells in culture, most of these cells showed inwardly rectifying K+-current. This current was significantly (4.5 ± 2.3 fold 12 of 12 cells) enhanced by application of Rb+ solution as this is a unique property of the RPE specific Kir7.1 channel. These cells tested positive for RPE specific transcripts.
This study demonstrated that commercially available Clonetics®human RPE cells from LONZA in RtEGMTM medium retain both molecular and electrophysiological phenotypes making them suitable for the study of disease mechanisms involving RPE. They can thus be used for the investigations of age-related macular degeneration, retinitis pigmentosa, determining gene expression profiles of RPE cells and potentially for subsequent transplantation studies.
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