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Cited by 401Retinal Diseases and TreatmentsRetinal Development and DisordersBarrier Structure and Function Studies

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue

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Maminishkis, Arvydas, Chen, Shan, Jalickee, Stephen, Banzon, Tina, Shi, Guangpu, Wang, Fei E., Ehalt, Todd, Hammer, Jeffrey A. +1 more·2006·Investigative Opthalmology & Visual Science
DOI 10.1167/iovs.05-1622PMC PMC1904392
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Abstract

PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.

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  • 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 Maminishkis et al., 2006

  • Human fetal RPE cultures have a high trans-epithelial electrical resistance (TER), from 500 to over 800 Ω cm2.

    “These cultures also have a high trans-epithelial electrical resistance (TER), from 500 to over 800 Ω cm2 (Hu and Bok, 2001; Maminishkis et al., 2006; Sonoda et al., 2009).”

    From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Maminishkis et al., 2006

  • There are now reproducible protocols that generate RPE monolayers with a well-differentiated phenotype for primary RPE cultures, RPE cells derived from stem cells, and ARPE-19 cells.

    “As discussed above, there are now reproducible protocols that generate RPE monolayers with a well-differentiated phenotype, not only for primary RPE cultures (Gibbs and Williams, 2003; Maminishkis et al., 2006; Strunnikova et al., 2010; Blenkinsop et al., 2013; Toops et al., 2014) and RPE cells derived from stem cells (Idelson et al., 2009; Buchholz et al., 2013; Gong et al., 2015; Maruotti et al.”

    From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Maminishkis et al., 2006

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Authors
Maminishkis, A., Chen, S., Jalickee, S., Banzon, T., Shi, G., Wang, F. E., Ehalt, T., Hammer, J. A., & Miller, S. S.
Affiliations
National Institutes of Health (US)
ORCID
Venue
Investigative Opthalmology & Visual Science
Publisher
Association for Research in Vision and Ophthalmology (ARVO)
Published
2006-08-01
Volume
47
Issue
8
Pages
3612
ISSN
  • 0146-0404 (ISSN-L)
  • 1552-5783
DOI
10.1167/iovs.05-1622
PMC
PMC1904392
OpenAlex
W1973207504
Cited by
401
References
97
Citations by year
2026: 11 · 2025: 7 · 2024: 14 · 2023: 20 · 2022: 23 · 2021: 18 · 2020: 27 · 2019: 22 · 2018: 25 · 2017: 24 · 2016: 21 · 2015: 34 · 2014: 30 · 2013: 24 · 2012: 25
FWCI
11.12
Type
journal article
Language
en
Open access
Locations
  • Investigative Ophthalmology & Visual Science (publishedVersion, public-domain)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (1z01ey000419-05)
  • National Eye Institute (Z01 EY000419-02)
Topics
  • Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
retinal pigment epithelium, primary cell culture, epithelial polarity, tight junctions, transepithelial potential, transepithelial electrical resistance, fluid absorption, ATP, VEGF secretion
MeSH terms
Pigment Epithelium-Derived Factor, Cell Separation, Electrophysiology, Enzyme-Linked Immunosorbent Assay, Eye Proteins, Fetus, Humans, Membrane Potentials, Microscopy, Electron, Nerve Growth Factors, Pigment Epithelium of Eye, Blotting, Western, Serpins, Cytokines, Cell Polarity, Cell Culture Techniques, Fluorescent Antibody Technique, Indirect, Vascular Endothelial Growth Factor A

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