Abstract
The blood-retinal barrier (BRB) present in the posterior chamber of the eye plays a major role in maintaining the proper function and integrity of the retina. Retinal pigment epithelium and choriocapillaris form the outer blood retinal barrier (oBRB), and breakdown of this barrier leads to vision-threatening diseases like macular edema, macular degeneration, and diabetic retinopathy. A simplified cell culture model of oBRB will be of great importance in elucidating the molecular mechanism of the disease progression. This chapter describes methods for primary cell isolation from donor eyes to culture human retinal pigment epithelial cells (hRPE) and choroidal endothelial cells (hCEC) and the protocol for construction of a simplified in vitro model of oBRB on fibronectin-coated Transwell inserts. Further, we explained the permeability study using FITC-dextran conjugated tracers for validating the bilayer model. The permeability experiments ensured that the system could easily be manipulated to recapitulate the pathological condition in vitro. Thus, it would be an optimal system for studying the disease mechanisms related to retinal and choroidal pathologies, for screening small molecules, and for performing drug permeability kinetics. Moreover, fundamental understanding of paracellular and transcellular trafficking of cargo in hRPE and hCEC could also be studied using this model.
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References
Simó R, Villarroel M, Corraliza L, et al (2010) The retinal pigment epithelium: something more than a constituent of the blood-retinal barrier-implications for the pathogenesis of diabetic retinopathy. J Biomed Biotechnol 1–15. https://doi.org/10.1155/2010/190724
Zhu M, Med M, Provis J, Penfold P (1998) Isolation, culture and characteristics of human foetal and adult retinal pigment epithelium. Aust New Zeal J Opthalmol 26(Suppl 1):S50–S52. Epub 1998/07/31
Kuznetsova A V., Kurinov AM, Aleksandrova MA (2014) Cell models to study regulation of cell transformation in pathologies of retinal pigment epithelium. J Ophthalmol 1–18. https://doi.org/10.1155/2014/801787
Cai H, Del Priore LV (2006) Gene expression profile of cultured adult compared to immortalized human RPE. Mol Vis 12:1–14
Mannermaa E, Reinisalo M, Ranta V-P et al (2010) Filter-cultured ARPE-19 cells as outer blood-retinal barrier model. Eur J Pharm Sci 40:289–296. https://doi.org/10.1016/j.ejps.2010.04.001
Chung M, Lee S, Lee BJ et al (2018) Wet-AMD on a chip: modeling outer blood-retinal barrier in vitro. Adv Healthc Mater 7:1–7. https://doi.org/10.1002/adhm.201700028
Maugeri G, D’Amico AG, Rasà DM et al (2017) Caffeine prevents blood retinal barrier damage in a model, in vitro, of diabetic macular edema. J Cell Biochem 118:2371–2379. https://doi.org/10.1002/jcb.25899
Spencer C, Abend S, McHugh KJ, Saint-Geniez M (2017) Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium. J Cell Mol Med 21:2542–2552. https://doi.org/10.1111/jcmm.13175
Browning AC, Halligan EP, Stewart EA et al (2012) Comparative gene expression profiling of human umbilical vein endothelial cells and ocular vascular endothelial cells. Br J Ophthalmol 96:128–132. https://doi.org/10.1136/bjophthalmol-2011-300572
Smith JR, Choi D, Chipps TJ et al (2007) Unique gene expression profiles of donor-matched human retinal and choroidal vascular endothelial cells. Investig Ophthalmol Vis Sci 48:2676–2684. https://doi.org/10.1167/iovs.06-0598
Palanisamy K, Karunakaran C, Raman R, Chidambaram S (2019) Optimization of an in vitro bilayer model for studying the functional interplay between human primary retinal pigment epithelial and choroidal endothelial cells isolated from donor eyes. BMC Res Notes 12. https://doi.org/10.1186/s13104-019-4333-x
Sonoda S, Sreekumar PG, Kase S et al (2010) Attainment of polarity promotes growth factor secretion by retinal pigment epithelial cells: relevance to age-related macular degeneration. Aging (Albany NY) 2:28–42
Ma X, Zhang H, Pan Q et al (2013) Hypoxia/aglycemia-induced endothelial barrier dysfunction and tight junction protein downregulation can be ameliorated by citicoline. PLoS One 8:4–9. https://doi.org/10.1371/journal.pone.0082604
Klaassen I, Van Noorden CJF, Schlingemann RO (2013) Molecular basis of the inner blood e retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog Retin Eye Res. https://doi.org/10.1016/j.preteyeres.2013.02.001
Acknowledgments
We would like to acknowledge the financial assistance provided by Department of Biotechnology under the project “DBT-RGYI BT/PR15023/GBD/27/282/2010,” DBT-Neurobiology Taskforce grant BT/PR15023/GBD/27/282/2010, and Max Planck-India mobility grant “IGSTC/MPG/FS (SC) 2012.” CS thanks the support from University Grants Commission, New Delhi, for the award of Assistant Professorship under its Faculty Recharge Program (UGC-FRP).
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Palanisamy, K., Chidambaram, S. (2024). An In Vitro Bilayer Model of Human Primary Retinal Pigment Epithelial and Choroid Endothelial Cells for Permeability Studies. In: Tharakan, B. (eds) Vascular Hyperpermeability. Methods in Molecular Biology, vol 2711. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3429-5_17
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DOI: https://doi.org/10.1007/978-1-0716-3429-5_17
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