Abstract
The cultivation of retinal pigment epithelium (RPE)-choroid explants gives the opportunity to study the RPE and Bruch’s membrane in its natural environment. Porcine eyes are easily available and an excellent model for human RPE. Explants are prepared less than 4 h postmortem from cooled eyes and are transferred in fixation rings. The tissues held between rings are cultured in a perfusion organ culture system for up to a week. Viability of the explants can be investigated by calcein staining.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Strauss O (2005) The retinal pigment epithelium in visual function. Physiol Rev 85:845–881
Combes RD, Shah AB (2016) The use of in vivo, ex vivo, in vitro, computational models and volunteer studies in vision research and therapy, and their contribution to the three Rs. Altern Lab Anim 44:187–238
Dithmer M, Fuchs S, Shi Y et al (2014) Fucoidan reduces secretion and expression of vascular endothelial growth factor in the retinal pigment epithelium and reduces angiogenesis in vitro. PLoS One 9:e89150
Guduric-Fuchs J, Ringland LJ, Gu P et al (2009) (2009) Immunohistochemical study of pig retinal development. Mol Vis 15:1915–1928
Lassota N (2008) Clinical and histological aspects of CNV formation: studies in an animal model. Acta Ophthalmol 86(Thesis 2):1–24
Sanchez I, Martin R, Ussa F et al (2011) The parameters of the porcine eyeball. Graefes Arch Clin Exp Ophthalmol 249:475–482
Middleton S (2010) Porcine ophthalmology. Vet Clin North Am Food Anim Pract 26:557–572
Matsumoto B, Guérin CJ, Anderson DH (1990) Cytoskeletal redifferentiation of feline, monkey, and human RPE cells in culture. Invest Ophthalmol Vis Sci 31:879–889
Grisanti S, Guidry C (1995) Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. Invest Ophthalmol Vis Sci 36:391–405
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
Burke JM (2008) Epithelial phenotype and the RPE: is the answer blowing in the Wnt? Prog Retin Eye Res 27:579–595
McKay BS, Irving PE, Skumatz CM et al (1997) Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells. Exp Eye Res 65:661–671
Kuriyama F, Ueda Y, Araki M (2009) Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices. Dev Neurobiol 69:950–958
Tamiya S, Liu L, Kaplan HJ (2010) Epithelial-mesenchymal transition and proliferation of retinal pigment epithelial cells initiated upon loss of cell-cell contact. Invest Ophthalmol Vis Sci 51:2755–2763
Treumer F, Klettner A, Baltz J et al (2012) Vectorial release of matrix metalloproteinases (MMPs) from porcine RPE-choroid explants following selective retina therapy (SRT): towards slowing the macular ageing process. Exp Eye Res 97:63–72
Miura Y, Klettner A, Noelle B et al (2010) Change of morphological and functional characteristics of retinal pigment epithelium cells during cultivation of retinal pigment epithelium-choroid perfusion tissue culture. Ophthalmic Res 43:122–133
Minuth WW, Stöckl G, Kloth S et al (1992) Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions. Eur J Cell Biol 57:132–137
Minuth WW, Kloth S, Aigner J et al (1996) Approach to an organo-typical environment for cultured cells and tissues. BioTechniques 20:498–501
Minuth WW, Schumacher K, Strehl R et al (2000) Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering. J Biomater Sci Polym Ed 11:495–522
Framme C, Kobuch K, Eckert E et al (2002) RPE in perfusion tissue culture and its response to laser application. Preliminary report. Ophthalmologica 216:320–328
Kobuch K, Herrmann WA, Framme C et al (2008) Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model. Exp Eye Res 86:661–668
Hammer M, Richter S, Kobuch K et al (2008) Intrinsic tissue fluorescence in an organotypic perfusion culture of the porcine ocular fundus exposed to blue light and free radicals. Graefes Arch Clin Exp Ophthalmol 246:979–988
Klettner A, Recber M, Roider J (2014) Comparison of the efficacy of aflibercept, ranibizumab, and bevacizumab in an RPE/choroid organ culture. Graefes Arch Clin Exp Ophthalmol 252:1593–1598
Klettner A, Westhues D, Lassen J et al (2013) Regulation of constitutive vascular endothelial growth factor secretion in retinal pigment epithelium/choroid organ cultures: p38, nuclear factor κB, and the vascular endothelial growth factor receptor-2/phosphatidylinositol 3 kinase pathway. Mol Vis 19:281–291
Johnson S, Rabinovitch P (2012) Ex vivo imaging of excised tissue using vital dyes and confocal microscopy. Curr Protoc Cytom Chapter 9:Unit 9.39
Pegg DE et al (1972) The choice of plastic tubing for organ perfusion experiments. Cryobiology 9:569–571
Acknowledgments
AK is funded by the Hermann-Wacker foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Klettner, A., Miura, Y. (2019). Porcine RPE/Choroidal Explant Cultures. In: Weber, B.H.F., Langmann, T. (eds) Retinal Degeneration. Methods in Molecular Biology, vol 1834. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8669-9_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8669-9_8
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-8668-2
Online ISBN: 978-1-4939-8669-9
eBook Packages: Springer Protocols