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 language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Pages | 45-65 |
| Number of pages | 21 |
| Volume | 945 |
| DOIs | |
| State | Published - Jan 1 2013 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 945 |
| ISSN (Print) | 10643745 |
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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
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 proceeding › Chapter
}
TY - CHAP
T1 - The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye
AU - Blenkinsop, Timothy A.
AU - Salero, Enrique
AU - Stern, Jeffrey H.
AU - Temple, Sally
PY - 2013/1/1
Y1 - 2013/1/1
N2 - 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.
AB - 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.
KW - Age-related macular degeneration
KW - Characterization of RPE
KW - Epithelial mesenchymal transition
KW - Human adult retinal pigment epithelium
KW - RPE culture
KW - RPE expansion
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UR - http://www.scopus.com/inward/citedby.url?scp=84870519702&partnerID=8YFLogxK
U2 - 10.1007/978-1-62703-125-7_4
DO - 10.1007/978-1-62703-125-7_4
M3 - Chapter
C2 - 23097100
AN - SCOPUS:84870519702
SN - 9781627031240
VL - 945
T3 - Methods in Molecular Biology
SP - 45
EP - 65
BT - Methods in Molecular Biology
ER -