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Heller JP, Kwok JC, Vecino E, et al. A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases. Front Cell Neurosci. 2015;9:449doi: 10.3389/fncel.2015.00449.
Heller, J. P., Kwok, J. C., Vecino, E., Martin, K. R., & Fawcett, J. W. (2015). A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases. Frontiers in cellular neuroscience, 9449. https://doi.org/10.3389/fncel.2015.00449
Heller, Janosch P, et al. "A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases." Frontiers in cellular neuroscience vol. 9 (2015): 449. doi: https://doi.org/10.3389/fncel.2015.00449
Heller JP, Kwok JC, Vecino E, Martin KR, Fawcett JW. A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases. Front Cell Neurosci. 2015 Nov 20;9:449. doi: 10.3389/fncel.2015.00449. eCollection 2015. PMID: 26635529; PMCID: PMC4654064.
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Front Cell Neurosci. 2015 Nov 20;9:449. doi: 10.3389/fncel.2015.00449. eCollection 2015.

A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases.

Frontiers in cellular neuroscience

Janosch P Heller, Jessica C F Kwok, Elena Vecino, Keith R Martin, James W Fawcett

Affiliations

  1. John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Cambridge, UK ; Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London London, UK.
  2. John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Cambridge, UK.
  3. John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Cambridge, UK ; Department of Cellular Biology, University of the Basque Country Leioa, UPV/EHU, Bizkaia, Spain.
  4. John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge Cambridge, UK ; Department of Ophthalmology, NIHR Biomedical Research Centre and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge Cambridge, UK.

PMID: 26635529 PMCID: PMC4654064 DOI: 10.3389/fncel.2015.00449

Abstract

Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 10(4) viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch's membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch's membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

Keywords: adult; age-related macular degeneration; culture; isolation; papain; rat; retina; retinal pigment epithelium

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