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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sonoda S, Spee C, Barron E, Ryan SJ, Kannan R, Hinton DR (2009) A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nat Protoc 4:662–673
Gamm DM, Melvan JN, Shearer RL, Pinilla I, Sabat G, Svendsen CN, Wright LS (2008) A novel serum-free method for culturing human prenatal retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 49:788–799
Geisen P, McColm JR, King BM, Hartnett ME (2006) 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
Maminishkis A, Chen S, Jalickee S, Banzon T, Shi G, Wang FE, Ehalt T, Hammer JA, Miller SS (2006) Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci 47:3612–3624
Flood MT, Gouras P (1981) The organization of human retinal pigment epithelium in vitro. Vis Res 21:119–126
Vielkind U, Crawford BJ (1988) Evaluation of different procedures for the dissociation of retinal pigmented epithelium into single viable cells. Pigment Cell Res 1:419–433
Lopashov GV (1983) Transdifferentiation of pigmented epithelium induced by the influence of lens epithelium in frogs. Differentiation 24:27–32
Grisanti S, Guidry C (1995) Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. Invest Ophthalmol Vis Sci 36:391–405
Casaroli-Marano RP, Pagan R, Vilaro S (1999) Epithelial–mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 40: 2062–2072
Saika S, Kono-Saika S, Tanaka T, Yamanaka O, Ohnishi Y, Sato M, Muragaki Y, Ooshima A, Yoo J, Flanders KC, Roberts AB (2004) Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice. Lab Invest 84:1245–1258
Lee H, O’Meara SJ, O’Brien C, Kane R (2007) The role of gremlin, a BMP antagonist, and epithelial-to-mesenchymal transition in proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci 48:4291–4299
Garcia S, Lopez E, Lopez-Colome AM (2008) Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms. J Cell Biochem 104:377–390
Kim JW, Kang KH, Burrola P, Mak TW, Lemke G (2008) Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium. Genes Dev 22:3147–3157
Liu Y, Ye F, Li Q, Tamiya S, Darling DS, Kaplan HJ, Dean DC (2009) Zeb1 represses Mitf and regulates pigment synthesis, cell proliferation, and epithelial morphology. Invest Ophthalmol Vis Sci 50:5080–5088
Tamiya S, Liu L, Kaplan HJ (2009) Loss of cell–cell contact initiates epithelial–mesenchymal transition and proliferation of retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 50(11):5080–5088
Liu Y, Xin Y, Ye F, Wang W, Lu Q, Kaplan HJ, Dean DC (2010) Taz-Tead1 Links Cell-Cell Contact to Zeb1 Expression, Proliferation and Dedifferen-tiation in Retinal Pigment Epithelial Cells. Invest Ophthalmol Vis Sci 51(7):3372–3378
Hay ED (1995) An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154:8–20
Tso MO, Cunha-Vaz JG, Shih CY, Jones CW (1980) Clinicopathologic study of blood-retinal barrier in experimental diabetes mellitus. Arch Ophthalmol 98:2032–2040
Caldwell RB, McLaughlin RJ, Boykins LG (1982) Intramembrane changes in retinal pigment epithelial cell junctions of the dystrophic rat retina. Invest Ophthalmol Vis Sci 23:305–318
Noell WK, Albrecht R (1971) Irreversible effects on visible light on the retina: role of vitamin A. Science 172:76–79
Bridges CD (1976) Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye. Exp Eye Res 22:435–455
Saari JC, Bredberg L, Garwin GG (1982) Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium. J Biol Chem 257:13329–13333
Edwards RB, Szamier RB (1977) Defective phagocytosis of isolated rod outer segments by RCS rat retinal pigment epithelium in culture. Science 197:1001–1003
Noell WK, Crapper DR, Paganelli CV (1965) Transretinal currents and ion fluxes. In: Snell FM, Noell WK (eds) Transcellular membrane potentials and ion fluxes. Gordon and Breach, New York, NY, pp 92–130
Steinberg RH, Miller S (1973) Aspects of electrolyte transport in frog pigment epithelium. Exp Eye Res 16:365–372
Miller SS, Steinberg RH (1977) Active transport of ions across frog retinal pigment epithelium. Exp Eye Res 25:235–248
Miller SS, Steinberg RH (1977) Passive ionic properties of frog retinal pigment epithelium. J Membr Biol 36:337–372
Eagle RC Jr (1984) Mechanisms of maculopathy. Ophthalmology 91:613–625
Burke JM, Skumatz CM, Irving PE, McKay BS (1996) Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ. Exp Eye Res 62:63–73
Feng W, Zheng JJ, Lutz DA, McLaughlin BJ (2003) Loss of RPE phenotype affects phagocytic function. Graefes Arch Clin Exp Ophthalmol 241:232–240
Whittaker JR (1967) Loss of melanotic phenotype in vitro by differentiated retinal pigment cells: demonstration of mechanisms involved. Dev Biol 15:553–574
Flood MT, Gouras P, Kjeldbye H (1980) Growth characteristics and ultrastructure of human retinal pigment epithelium in vitro. Invest Ophthalmol Vis Sci 19:1309–1320
Rodriguez-Boulan E, Nelson WJ (1989) Morphogenesis of the polarized epithelial cell phenotype. Science 245:718–725
Luna EJ, Hitt AL (1992) Cytoskeleton–plasma membrane interactions. Science 258:955–964
Hitt AL, Luna EJ (1994) Membrane interactions with the actin cytoskeleton. Curr Opin Cell Biol 6:120–130
Turksen K, Opas M, Kalnins VI (1989) Cytoskeleton, adhesion, and extracellular matrix of fetal human retinal pigmented epithelial cells in culture. Ophthalmic Res 21:56–66
Song MK, Lui GM (1990) Propagation of fetal human RPE cells: preservation of original culture morphology after serial passage. J Cell Physiol 143:196–203
Gouras P, Cao H, Sheng Y, Tanabe T, Efremova Y, Kjeldbye H (1994) Patch culturing and transfer of human fetal retinal epithelium. Graefes Arch Clin Exp Ophthalmol 232: 599–607
Castillo BV Jr, Little CW, del Cerro C, del Cerro M (1995) An improved method of isolating fetal human retinal pigment epithelium. Curr Eye Res 14:677–683
Zhu M, Provis JM, Penfold PL (1998) Isolation, culture and characteristics of human foetal and adult retinal pigment epithelium. Aust N Z J Ophthalmol 26(Suppl 1):S50–S52
Hu J, Bok D (2001) A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers. Mol Vis 7:14–19
Rak DJ, Hardy KM, Jaffe GJ, McKay BS (2006) Ca++-switch induction of RPE differentiation. Exp Eye Res 82:648–656
Tezel TH, Del Priore LV (1998) Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium. Exp Eye Res 66:807–815
McKay BS, Burke JM (1994) Separation of phenotypically distinct subpopulations of cultured human retinal pigment epithelial cells. Exp Cell Res 213:85–92
Stump RJ, Lovicu FJ, Ang SL, Pandey SK, McAvoy JW (2006) Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition. J Pathol 210:249–257
Papageorgis P, Lambert AW, Ozturk S, Gao F, Pan H, Manne U, Alekseyev YO, Thiagalingam A, Abdolmaleky HM, Lenburg M, Thiagalingam S (2010) Smad signaling is required to maintain epigenetic silencing during breast cancer progression. Cancer Res 70:968–978
De S, Rabin DM, Salero E, Lederman PL, Temple S, Stern JH (2007) Human retinal pigment epithelium cell changes and expression of alphaB-crystallin: a biomarker for retinal pigment epithelium cell change in age-related macular degeneration. Arch Ophthalmol 125:641–645
Hemesath TJ, Steingrimsson E, McGill G, Hansen MJ, Vaught J, Hodgkinson CA, Arnheiter H, Copeland NG, Jenkins NA, Fisher DE (1994) Microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family. Genes Dev 8:2770–2780
McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, Schneeberger EE (1996) Occludin is a functional component of the tight junction. J Cell Sci 109(Pt 9):2287–2298
Redmond TM, Yu S, Lee E, Bok D, Hamasaki D, Chen N, Goletz P, Ma JX, Crouch RK, Pfeifer K (1998) Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet 20:344–351
Denker BM, Nigam SK (1998) Molecular structure and assembly of the tight junction. Am J Physiol 274:F1–F9
Martinez-Morales JR, Dolez V, Rodrigo I, Zaccarini R, Leconte L, Bovolenta P, Saule S (2003) OTX2 activates the molecular network underlying retina pigment epithelium differentiation. J Biol Chem 278:21721–21731
Saari JC, Nawrot M, Kennedy BN, Garwin GG, Hurley JB, Huang J, Possin DE, Crabb JW (2001) Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation. Neuron 29:739–748
Fischmeister R, Hartzell HC (2005) Volume sensitivity of the bestrophin family of chloride channels. J Physiol 562:477–491
Hunt RC, Davis AA (1990) Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro. J Cell Physiol 145:187–199
Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–169
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Blenkinsop, T.A., Salero, E., Stern, J.H., Temple, S. (2012). The Culture and Maintenance of Functional Retinal Pigment Epithelial Monolayers from Adult Human Eye. In: Randell, S., Fulcher, M. (eds) Epithelial Cell Culture Protocols. Methods in Molecular Biology, vol 945. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-125-7_4
Download citation
DOI: https://doi.org/10.1007/978-1-62703-125-7_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-124-0
Online ISBN: 978-1-62703-125-7
eBook Packages: Springer Protocols