Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells

Abstract

We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Diagram of the outer retina illustrating the polarized nature of the RPE monolayer and its relationship to the photoreceptor inner and outer segments, Bruch's membrane and the choriocapillaris.
Figure 2: Human fetal eye with the first incision.
Figure 3: The fetal eye is split into cornea–iris (anterior segment), lens and posterior eye cup.
Figure 4: The human fetal eye is dissected into 4 quadrants.
Figure 5: The RPE–choroid layer is peeled off from the sclera using forceps.
Figure 6: Intact RPE sheets (black arrows) are peeled from the choroid with fine forceps under a dissecting microscope.
Figure 7: Light microscopic images of early passage confluent fetal RPE cells grown on a plastic tissue culture plate.
Figure 8: Light microscopic images showing pattern of time-dependent growth of normal RPE in a T75 flask and highly pigmented and overgrown multi-layers of RPE for comparison.
Figure 9
Figure 10: Time dependent increase in TER from multiple Transwells containing RPE from a single donor.
Figure 11: Evidence for differentiation and polarization in human fetal RPE cells cultured on Transwell filters for 4 weeks.
Figure 12: Steps involved in the isolation of pure RPE sheets.
Figure 13: Measurment of TER of polarized human RPE using a voltohmmeter.

Similar content being viewed by others

Zixuan Zhao, Xinyi Chen, … Hanry Yu

References

  1. Thumann, G., Hoffmann, S. & Hinton, D.R. Cell biology of the retinal pigment epithelium. In Retina. 4th edn. Vol. 1 (ed. Ryan, S.J.) 137–152 (Elsevier Mosby, Philadelphia, USA, 2006).

    Chapter  Google Scholar 

  2. Rodriguez-Boulan, E. & Nelson, W.J. Morphogenesis of the polarized epithelial cell phenotype. Science 245, 718–725 (1989).

    Article  CAS  Google Scholar 

  3. Marmor, M.F., Abdul-Rahim, A.S. & Cohen, D.S. The effect of metabolic inhibitors on retinal adhesion and subretinal fluid resorption. Invest. Ophthalmol. Vis. Sci. 19, 893–903 (1980).

    CAS  PubMed  Google Scholar 

  4. Miller, S.S., Hughes, B.A. & Machen, T.E. Fluid transport across retinal pigment epithelium is inhibited by cyclic AMP. Proc. Natl Acad. Sci. USA 79, 2111–2115 (1982).

    Article  CAS  Google Scholar 

  5. Anderson, J.M. & Van Itallie, C.M. Tight junctions and the molecular basis for regulation of paracellular permeability. Am. J. Physiol. 269, G467–G475 (1995).

    CAS  PubMed  Google Scholar 

  6. Rizzolo, L.J. Polarity and the development of the outer blood-retinal barrier. Histol. Histopathol. 12, 1057–1067 (1997).

    CAS  PubMed  Google Scholar 

  7. Hogan, M.J. Role of the retinal pigment epithelium in macular disease. Trans. Am. Acad. Ophthalmol. Otolaryngol. 76, 64–80 (1972).

    CAS  PubMed  Google Scholar 

  8. Sheedlo, H.J., Li, L. & Turner, J.E. Effects of RPE-cell factors secreted from permselective fibers on retinal cells in vitro . Brain Res. 587, 327–337 (1992).

    Article  CAS  Google Scholar 

  9. Holtkamp, G.M. et al. Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells. Clin. Exp. Immunol. 112, 34–43 (1998).

    Article  CAS  Google Scholar 

  10. Blaauwgeers, H.G. et al. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am. J. Pathol. 155, 421–428 (1999).

    Article  CAS  Google Scholar 

  11. Hu, J. & Bok, D. A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers. Mol. Vis. 7, 14–19 (2001).

    CAS  PubMed  Google Scholar 

  12. Maminishkis, A. et al. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest. Ophthalmol. Vis. Sci. 47, 3612–3624 (2006).

    Article  Google Scholar 

  13. Dunn, K.C., Aotaki-Keen, A.E., Putkey, F.R. & Hjelmeland, L.M. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp. Eye Res. 62, 155–169 (1996).

    Article  CAS  Google Scholar 

  14. Kannan, R. et al. Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells. Mol. Vis. 12, 1649–1659 (2006).

    CAS  PubMed  Google Scholar 

  15. Geisen, P., McColm, J.R., King, B.M. & Hartnett, M.E. Characterization of barrier properties and inducible VEGF expression of several types of retinal pigment epithelium in medium-term culture. Curr. Eye Res. 31, 739–748 (2006).

    Article  CAS  Google Scholar 

  16. Okami, T. et al. Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells. J. Histochem. Cytochem. 38, 1267–1275 (1990).

    Article  CAS  Google Scholar 

  17. Stevenson, B.R., Siliciano, J.D., Mooseker, M.S. & Goodenough, D.A. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol. 103, 755–766 (1986).

    Article  CAS  Google Scholar 

  18. Quinn, R.H. & Miller, S.S. Ion transport mechanisms in native human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 33, 3513–3527 (1992).

    CAS  PubMed  Google Scholar 

  19. Philp, N.J., Wang, D., Yoon, H. & Hjelmeland, L.M. Polarized expression of monocarboxylate transporters in human retinal pigment epithelium and ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 44, 1716–1721 (2003).

    Article  Google Scholar 

  20. Ohno-Matsui, K. et al. Novel mechanism for age-related macular degeneration: an equilibrium shift between the angiogenesis factors VEGF and PEDF. J. Cell Physiol. 189, 323–333 (2001).

    Article  CAS  Google Scholar 

  21. Shi, G. et al. Control of chemokine gradients by the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 49, 4620–4630 (2008).

    Article  Google Scholar 

  22. Sonoda, S. et al. Selective increase in basolateral secretion of VEGF upon BMP-4 treatment of highly polarized human RPE cells. Invest. Ophthalmol. Vis. Sci. 49, ARVO E-Abstract 855 (2008).

  23. Zhang, N. et al. Characterization of brimonidine transport in retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 47, 287–294 (2006).

    Article  Google Scholar 

  24. Sreekumar, P.G. et al. N-(4-hydroxyphenyl) retinamide augments laser-induced choroidal neovascularization in mice. Invest. Ophthalmol. Vis. Sci. 49, 1210–1220 (2008).

    Article  Google Scholar 

  25. German, O.L., Buzzi, E., Rotstein, N.P., Rodriguez-Boulan, E. & Politi, L.E. Retinal pigment epithelial cells promote spatial reorganization and differentiation of retina photoreceptors. J. Neurosci. Res. 86, 3503–3514 (2008).

    Article  CAS  Google Scholar 

  26. Senanayake, P. et al. Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface. J. Cell Sci. 114, 199–205 (2001).

    CAS  Google Scholar 

  27. Hamel, C.P. et al. A developmentally regulated microsomal protein specific for the pigment epithelium of the vertebrate retina. J. Neurosci. Res. 34, 414–425 (1993).

    Article  CAS  Google Scholar 

  28. Jin, M., Barron, E., He, S., Ryan, S.J. & Hinton, D.R. Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor. Invest. Ophthalmol. Vis. Sci. 43, 2782–2790 (2002).

    PubMed  Google Scholar 

  29. Tandler, B. Improved uranyl acetate staining for electron microscopy. J. Electron. Microsc. Tech. 16, 81–82 (1990).

    Article  CAS  Google Scholar 

  30. Ban, Y. & Rizzolo, L.J. A culture model of development reveals multiple properties of RPE tight junctions. Mol. Vis. 3, 18 (1997).

    CAS  PubMed  Google Scholar 

  31. Frambach, D.A., Fain, G.L., Farber, D.B. & Bok, D. Beta adrenergic receptors on cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 31, 1767–1772 (1990).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Supported by funds from The Arnold and Mabel Beckman Foundation, National Institutes of Health Grants EY01545 and core grant EY03040, and the Research to Prevent Blindness Inc. The authors thank P.G. Sreekumar for his early contributions to the development of these methods.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to David R Hinton.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sonoda, S., Spee, C., Barron, E. et al. A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nat Protoc 4, 662–673 (2009). https://doi.org/10.1038/nprot.2009.33

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.33

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing