The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye

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

Research output: Chapter in Book/Report/Conference proceedingChapter

45 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages45-65
Number of pages21
Volume945
DOIs
StatePublished - Jan 1 2013

Publication series

NameMethods in Molecular Biology
Volume945
ISSN (Print)10643745

Fingerprint

Retinal Pigments
Retinal Pigment Epithelium
Maintenance
Immunohistochemistry
Tissue Donors
Polymerase Chain Reaction
Eye Diseases
Cell Transplantation
Macular Degeneration
Cell Survival
Cell Death
Cell Culture Techniques
RNA

Keywords

  • Age-related macular degeneration
  • Characterization of RPE
  • Epithelial mesenchymal transition
  • Human adult retinal pigment epithelium
  • RPE culture
  • RPE expansion

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Blenkinsop, T. A., Salero, E., Stern, J. H., & Temple, S. (2013). The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye. In Methods in Molecular Biology (Vol. 945, pp. 45-65). (Methods in Molecular Biology; Vol. 945). https://doi.org/10.1007/978-1-62703-125-7_4

The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye. / Blenkinsop, Timothy A.; Salero, Enrique; Stern, Jeffrey H.; Temple, Sally.

Methods in Molecular Biology. Vol. 945 2013. p. 45-65 (Methods in Molecular Biology; Vol. 945).

Research output: Chapter in Book/Report/Conference proceedingChapter

Blenkinsop, TA, Salero, E, Stern, JH & Temple, S 2013, The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye. in Methods in Molecular Biology. vol. 945, Methods in Molecular Biology, vol. 945, pp. 45-65. https://doi.org/10.1007/978-1-62703-125-7_4
Blenkinsop TA, Salero E, Stern JH, Temple S. The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye. In Methods in Molecular Biology. Vol. 945. 2013. p. 45-65. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-62703-125-7_4
Blenkinsop, Timothy A. ; Salero, Enrique ; Stern, Jeffrey H. ; Temple, Sally. / The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye. Methods in Molecular Biology. Vol. 945 2013. pp. 45-65 (Methods in Molecular Biology).
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