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The Culture and Maintenance of Functional Retinal Pigment Epithelial Monolayers from Adult Human Eye

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Epithelial Cell Culture Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 945))

Abstract

The retinal pigment epithelium (RPE) is implicated in many eye diseases, including age-related macular degeneration, and therefore isolating and culturing these cells from recently deceased adult human donors is the ideal source for disease studies. Adult RPE could also be used as a cell source for transplantation therapy for RPE degenerative disease, likely requiring first in vitro expansion of the cells obtained from a patient. Previous protocols have successfully extracted RPE from adult donors; however improvements in yield, cell survival, and functionality are needed. We describe here a protocol optimized for adult human tissue that yields expanded cultures of RPE with morphological, phenotypic, and functional characteristics similar to freshly isolated RPE. These cells can be expanded and cultured for several months without senescence, gross cell death, or undergoing morphological changes. The protocol takes around a month to obtain functional RPE monolayers with accurate morphological characteristics and normal protein expression, as shown through immunohistochemistry analysis, RNA expression profiles via quantitative PCR (qPCR), and transepithelial resistance (TER) measurements. Included in this chapter are steps used to extract RPE from human adult globes, cell culture, cell splitting, cell bleaching, immunohistochemistry, and qPCR for RPE markers, and TER measurements as functional test.

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Authors and Affiliations

  1. Neural Stem Cell Institute, Rensselaer, NY, USA

    Timothy A. Blenkinsop, Enrique Salero, Jeffrey H. Stern & Sally Temple

Authors
  1. Timothy A. Blenkinsop
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  2. Enrique Salero
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  3. Jeffrey H. Stern
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  4. Sally Temple
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Corresponding author

Correspondence to Sally Temple .

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Editors and Affiliations

  1. Cystic Fibrosis/Pulmonary Research Ctr., University of North Carolina, Thurston Bowles Building 4011, Chapel Hill, 27599, North Carolina, USA

    Scott H. Randell

  2. Cystic Fibrosis/Pulmonary Research Ctr., The University of North Carolina, Thurston-Bowles Building 4011, Chapel Hill, 27599, North Carolina, USA

    M. Leslie Fulcher

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Blenkinsop, T.A., Salero, E., Stern, J.H., Temple, S. (2012). The Culture and Maintenance of Functional Retinal Pigment Epithelial Monolayers from Adult Human Eye. In: Randell, S., Fulcher, M. (eds) Epithelial Cell Culture Protocols. Methods in Molecular Biology, vol 945. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-125-7_4

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  • DOI: https://doi.org/10.1007/978-1-62703-125-7_4

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-124-0

  • Online ISBN: 978-1-62703-125-7

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