The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye
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.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Protocols to develop cultures from adult human donors yield highly polarized RPE monolayers after at least 4 weeks in culture.
“Detailed protocols to develop cultures from adult human donors have been established and are reported to yield highly polarized RPE monolayers after at least 4 weeks in culture (Blenkinsop et al., 2013).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Blenkinsop et al., 2013
Primary RPE, iPS-RPE, and ES-RPE cells can form well-differentiated, RPE-like cultures, demonstrating a polarized epithelial organization, close approximation of native RPE gene and protein expression, and the ability to perform RPE functions such as ingestion and degradation of OSs over a time-course comparable to in vivo.
“An important characteristic of primary RPE, iPS-RPE, and ES-RPE cells is that they can form well-differentiated, RPE-like cultures, demonstrating a polarized epithelial organization (Fig. 2), close approximation of native RPE gene and protein expression, and the ability to perform RPE functions, such as the ingestion and degradation of OSs over a time-course that is comparable to that in vivo (Mam”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Blenkinsop et al., 2013
Cites
An Overview of Epithelio-Mesenchymal Transformation
Hay, E. D. (1995) · Cells Tissues Organs · doi:10.1159/000147748 · Cited by 1,577 · see citing works
ARPE-19, A Human Retinal Pigment Epithelial Cell Line with Differentiated Properties
Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., & Hjelmeland, L. M. (1996) · Experimental Eye Research · doi:10.1006/exer.1996.0020 · Cited by 1,334 · see citing works
Morphogenesis of the Polarized Epithelial Cell Phenotype
Rodriguez-Boulan, E. J., & Nelson, W. J. (1989) · Science · doi:10.1126/science.2672330 · Cited by 1,085 · see citing works
Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle
Redmond., T. M., Yu, S., Lee, E., Bok, D., Hamasaki, D., Chen, N., Goletz, P., Ma, J.-X., Crouch, R. K., & Pfeifer, K. (1998) · Nature Genetics · doi:10.1038/3813 · Cited by 929 · see citing works
Cytoskeleton—Plasma Membrane Interactions
Luna, E. J., & Hitt, A. L. (1992) · Science · doi:10.1126/science.1439807 · Cited by 786 · see citing works
Hemesath, T. J., Steingrímsson, E., McGill, G., Hansen, M. J., Vaught, J., Hodgkinson, C. A., Arnheiter, H., Copeland, N. G., Jenkins, N. A., & Fisher, D. E. (1994) · Genes & Development · doi:10.1101/gad.8.22.2770 · Cited by 673 · see citing works
Occludin is a functional component of the tight junction
McCarthy, K. M., Skare, I. B., Stankewich, M. C., Furuse, M., Tsukita, S., Rogers, R. A., Lynch, R. D., & Schneeberger, E. E. (1996) · Journal of Cell Science · doi:10.1242/jcs.109.9.2287 · Cited by 640 · see citing works
Molecular structure and assembly of the tight junction
Denker, B. M., & Nigám, S. K. (1998) · American Journal of Physiology-Renal Physiology · doi:10.1152/ajprenal.1998.274.1.f1 · Cited by 405 · see citing works
Maminishkis, A., Chen, S., Jalickee, S., Banzon, T., Shi, G., Wang, F. E., Ehalt, T., Hammer, J. A., & Miller, S. S. (2006) · Investigative Opthalmology & Visual Science · doi:10.1167/iovs.05-1622 · Cited by 401 · see citing works
Sonoda, S., Spee, C., Barron, E., Ryan, S. J., Kannan, R., & Hinton, D. R. (2009) · Nature Protocols · doi:10.1038/nprot.2009.33 · Cited by 272 · see citing works
Saari, J. C., Nawrot, M., Kennedy, B. N., Garwin, G. G., Hurley, J. B., Huang, J., Possin, D. E., & Crabb, J. W. (2001) · Neuron · doi:10.1016/s0896-6273(01)00248-3 · Cited by 250 · see citing works
Edwards, R. B., & Szamier, R. B. (1977) · Science · doi:10.1126/science.560718 · Cited by 250 · see citing works
Irreversible Effects of Visible Light on the Retina: Role of Vitamin A
Noell, W. K., & Albrecht, R. (1971) · Science · doi:10.1126/science.172.3978.76 · Cited by 248 · see citing works
(1995) · PubMed · Cited by 239 · see citing works
Passive ionic properties of frog retinal pigment epithelium
Miller, S. S., & Steinberg, R. H. (1977) · The Journal of Membrane Biology · doi:10.1007/bf01868158 · Cited by 206 · see citing works
Saari, J. C., Bredberg, L., & Garwin, G. G. (1982) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)33451-3 · Cited by 200 · see citing works
Growth characteristics and ultrastructure of human retinal pigment epithelium in vitro.
Flood, M. T., Gouras, P., & Kjeldbye, H. M. (1980) · PubMed · Cited by 187 · see citing works
OTX2 Activates the Molecular Network Underlying Retina Pigment Epithelium Differentiation
Martínez‐Morales, J. R., Dolez, V., Rodrigo, I., et al. (2003) · Journal of Biological Chemistry · doi:10.1074/jbc.m301708200 · Cited by 184 · see citing works
Smad Signaling Is Required to Maintain Epigenetic Silencing during Breast Cancer Progression
Papageorgis, P., Lambert, A. W., Ozturk, S., Gao, F., Pan, H., Manne, U., Alekseyev, Y. O., Thiagalingam, A., Abdolmaleky, H. M., Lenburg, M. E., et al. (2010) · Cancer Research · doi:10.1158/0008-5472.can-09-1872 · Cited by 177 · see citing works
Bridges, C. D. B. (1976) · Experimental Eye Research · doi:10.1016/0014-4835(76)90182-2 · Cited by 165 · see citing works
Hu, J. G., & Bok, D. (2001) · PubMed · Cited by 165 · see citing works
Casaroli-Marano, R. P., Pagan, R., & Vilaró, S. (1999) · PubMed · Cited by 164 · see citing works
Active transport of ions across frog retinal pigment epithelium
Miller, S. S., & Steinberg, R. H. (1977) · Experimental Eye Research · doi:10.1016/0014-4835(77)90090-2 · Cited by 164 · see citing works
One hundred years of journal publication
Appel, T. A. (1998) · American Journal of Physiology-Cell Physiology · doi:10.1152/ajpcell.1998.274.1.c1 · Cited by 164 · see citing works
Saika, S., Kono-Saika, S., Tanaka, T., et al. (2004) · Laboratory Investigation · doi:10.1038/labinvest.3700156 · Cited by 156 · see citing works
Membrane interactions with the actin cytoskeleton
Hitt, A. L., & Luna, E. J. (1994) · Current Opinion in Cell Biology · doi:10.1016/0955-0674(94)90125-2 · Cited by 136 · see citing works
Aspects of electrolyte transport in frog pigment epithelium
Steinberg, R. H., & Miller, S. S. (1973) · Experimental Eye Research · doi:10.1016/0014-4835(73)90130-9 · Cited by 126 · see citing works
Volume sensitivity of the bestrophin family of chloride channels
Fischmeister, R., & Hartzell, H. C. (2004) · The Journal of Physiology · doi:10.1113/jphysiol.2004.075622 · Cited by 123 · see citing works
Clinicopathologic Study of Blood-Retinal Barrier in Experimental Diabetes Mellitus
Tso, M. O. M., Cunha-Vaz, J. G., Shih, C.-Y., et al. (1980) · Archives of Ophthalmology · doi:10.1001/archopht.1980.01020040884020 · Cited by 107 · see citing works
Song, M., & Lui, G. M. (1990) · Journal of Cellular Physiology · doi:10.1002/jcp.1041430127 · Cited by 98 · see citing works
Eagle, R. C. (1984) · Ophthalmology · doi:10.1016/s0161-6420(84)34259-2 · Cited by 97 · see citing works
Lee, H., O'Meara, S. J., O'Brien, C. J., & Kane, R. (2007) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.07-0086 · Cited by 93 · see citing works
Hunt, R. C., & Davis, A. A. (1990) · Journal of Cellular Physiology · doi:10.1002/jcp.1041450202 · Cited by 88 · see citing works
Phenotypic Heterogeneity of Retinal Pigment Epithelial Cells In Vitro and In Situ
Burke, J. M., Skumatz, C. M. B., Irving, P. E., & McKay, B. S. (1996) · Experimental Eye Research · doi:10.1006/exer.1996.0008 · Cited by 84 · see citing works
Human Retinal Pigment Epithelium Cell Changes and Expression of αB-Crystallin
De, S. (2007) · Archives of Ophthalmology · doi:10.1001/archopht.125.5.641 · Cited by 80 · see citing works
Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epitheliumPreprint
Kim, J. W., Kang, K. H., Burrola, P., Mak, T. W., & Lemke, G. (2008) · Genes & Development · doi:10.1101/gad.1700108 · Cited by 80 · see citing works
Liu, Y., Xin, Y., Ye, F., Wang, W., Lu, Q., Kaplan, H. J., & Dean, D. C. (2010) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.09-4321 · Cited by 77 · see citing works
Geisen, P., McColm, J. R., King, B., & Hartnett, M. E. (2006) · Current Eye Research · doi:10.1080/02713680600837408 · Cited by 74 · see citing works
A Novel Serum-Free Method for Culturing Human Prenatal Retinal Pigment Epithelial Cells
Gamm, D. M., Melvan, J. N., Shearer, R. L., Pinilla, I., Sabat, G., Svendsen, C. N., & Wright, L. S. (2008) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.07-0777 · Cited by 72 · see citing works
McKay, B. S., & Burke, J. M. (1994) · Experimental Cell Research · doi:10.1006/excr.1994.1176 · Cited by 69 · see citing works
Zeb1 Represses Mitf and Regulates Pigment Synthesis, Cell Proliferation, and Epithelial Morphology
Liu, Y., Ye, F., Li, Q., Tamiya, S., Darling, D. S., Kaplan, H. J., & Dean, D. C. (2009) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.08-2911 · Cited by 60 · see citing works
Whittaker, J. R. (1967) · Developmental Biology · doi:10.1016/0012-1606(67)90053-x · Cited by 60 · see citing works
Stump, R. J. W., Lovicu, F. J., Ang, S. L., et al. (2006) · The Journal of Pathology · doi:10.1002/path.2049 · Cited by 43 · see citing works
Ca++-switch induction of RPE differentiation
Rak, D. J., Hardy, K. M., Jaffe, G. J., & McKay, B. S. (2005) · Experimental Eye Research · doi:10.1016/j.exer.2005.09.002 · Cited by 36 · see citing works
García, S. Z., López, E., & López-Colomé, A. M. (2007) · Journal of Cellular Biochemistry · doi:10.1002/jcb.21633 · Cited by 33 · see citing works
Serum-free Media for Culturing and Serial-Passaging of Adult Human Retinal Pigment Epithelium
Tezel, T. H., & Priore, L. V. D. (1998) · Experimental Eye Research · doi:10.1006/exer.1998.0492 · Cited by 30 · see citing works
Intramembrane changes in retinal pigment epithelial cell junctions of the dystrophic rat retina.
Caldwell, R. B., McLaughlin, R. J., & Boykins, L. G. (1982) · PubMed · Cited by 29 · see citing works
Patch culturing and transfer of human fetal retinal epithelium
Gouras, P., Cao, H., 盛耀华, Tanabe, T., Efremova, Y., & Kjeldbye, H. M. (1994) · Graefe s Archive for Clinical and Experimental Ophthalmology · doi:10.1007/bf00193120 · Cited by 29 · see citing works
The organization of human retinal pigment epithelium in vitro
Flood, M. T., & Gouras, P. (1981) · Vision Research · doi:10.1016/0042-6989(81)90144-9 · Cited by 27 · see citing works
Turksen, K., Opas, M., & Kalnins, V. I. (2009) · Ophthalmic Research · doi:10.1159/000266768 · Cited by 24 · see citing works
Isolation, culture and characteristics of human foetal and adult retinal pigment epithelium
Zhu, M., Provis, J. M., & Penfold, P. (1998) · Australian and New Zealand Journal of Ophthalmology · doi:10.1111/j.1442-9071.1998.tb01371.x · Cited by 19 · see citing works
An improved method of isolating fetal human retinal pigment epithelium
Castillo, B. V., Little, C. W., del Cerro, C., et al. (1995) · Current Eye Research · doi:10.3109/02713689508998495 · Cited by 17 · see citing works
Loss of RPE phenotype affects phagocytic function
Feng, W., Zheng, J. J., Lutz, D. A., & McLaughlin, B. J. (2003) · Graefe s Archive for Clinical and Experimental Ophthalmology · doi:10.1007/s00417-002-0617-1 · Cited by 15 · see citing works
Vielkind, U., & Crawford, B. J. (1988) · Pigment Cell Research · doi:10.1111/j.1600-0749.1988.tb00146.x · Cited by 15 · see citing works
Transdifferentiation of Pigmented Epithelium Induced by the Influence of Lens Epithelium in Frogs
Lopashov, G. V. (1983) · Differentiation · doi:10.1111/j.1432-0436.1983.tb01298.x · Cited by 10 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
The cell biology of the retinal pigment epithelium
Lakkaraju, A., Umapathy, A., Tan, L. X., Daniele, L., Philp, N. J., Boesze-Battaglia, K., & Williams, D. S. (2020) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2020.100846 · Cited by 422 · see citing works
Plasma membrane protein polarity and trafficking in RPE cells: past, present and future
Lehmann, G. L., Benedicto, I., Philp, N. J., & Rodriguez-Boulan, E. (2014) · Experimental Eye Research · doi:10.1016/j.exer.2014.04.021 · Cited by 122 · see citing works
Kaur, G., Tan, L. X., Rathnasamy, G., La Cunza, N., Germer, C. J., Toops, K. A., Fernandes, M., Blenkinsop, T. A., & Lakkaraju, A. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1805039115 · Cited by 79 · see citing works
Self-citationToops, K. A., Tan, L. X., & Lakkaraju, A. (2014) · Experimental Eye Research · doi:10.1016/j.exer.2014.05.003 · Cited by 73 · see citing works
Müller, C., Charniga, C., Temple, S., & Finnemann, S. C. (2018) · Stem Cell Reports · doi:10.1016/j.stemcr.2018.01.017 · Cited by 61 · see citing works
Self-citationParikh, B. H., Liu, Z., Blakeley, P., Lin, Q., Singh, M., Ong, J. Y., Ho, K. H., Lai, J. W., Bogireddi, H., Tran, K. C., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-30474-6 · Cited by 54 · see citing works
Self-citation
- Authors
- Blenkinsop, T. A., Salero, E., Stern, J. H., & Temple, S.
- Affiliations
- Neural Stem Cell Institute (US)
- Corresponding author
- Temple, Sally
- Venue
- Methods in molecular biology
- Publisher
- Humana Press
- Published
- 2012
- Volume
- 945
- Pages
- 45-65
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W46824281
- Cited by
- 96
- References
- 55
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 5 · 2023: 3 · 2022: 12 · 2021: 7 · 2020: 9 · 2019: 9 · 2018: 7 · 2017: 9 · 2016: 8 · 2015: 10 · 2014: 8 · 2013: 2
- FWCI
- 29.16
- Type
- book chapter
- Language
- en
- Open access
- closed
- Licence
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Retinal Imaging and Analysis
- Keywords
- retinal pigment epithelium, cell expansion, transepithelial electrical resistance, age-related macular degeneration, RPE transplantation, cellular senescence
- MeSH terms
- Adult, Collagen, Dissection, Drug Combinations, Humans, Immunohistochemistry, Laminin, Proteoglycans, Sucrose, Cell Culture Techniques, Retinal Pigment Epithelium, Transcriptome
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
