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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

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Biology

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Published: February 26th, 2021

DOI:

10.3791/62145

1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Department of Ophthalmology, University Hospital RWTH Aachen, 4Max Delbrück Center for Molecular Medicine
* These authors contributed equally

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo analyses and in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Our group is focusing on the development of regenerative approaches to treat neuroretinal degeneration, i.e., AMD, by RPE- and IPE-based non-viral gene therapy. The pre-clinical establishment of such therapies necessitates in vitro models transferable to human beings. Thus, the goal of the study presented here is to deliver protocols for the isolation, culture, and genetic engineering of primary RPE and IPE cells. The rationale to establish the isolation of PE cells from multiple species is to robustly confirm safety and efficiency of the approach and increase its reproducibility and transferability. The available human RPE cell line ARPE-19 differs from prim....

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The protocols in which animals were involved were carried out by certified personnel and after authorization by the cantonal Département de la sécurité, de l'emploi et de la santé (DSES), Domaine de l'expérimentation animale of Geneva, Switzerland, and according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (approval no. GE/94/17). Adult healthy Brown Norway rats, C57BL/6 mice, and New Zealand white rabbits were euthanized by an overdose of Pentobarbital (.......

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PE isolation from different mammal species
Using the aforementioned protocols, IPE and RPE cells were successfully isolated and cultured from five different species. The number of cells obtained from each procedure depends on the species and size of the eye (Table 1). As shown in Figure 1, cells show typical PE cell morphology and pigmentation (except for rabbit cells shown, derived from albino New Zealand White (NZW) rabbits). At 21 days post-isolatio.......

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Having standardized methods to isolate and culture PE cells is fundamental in developing new therapy approaches for retinal degenerative diseases. With the protocols presented here, PE cells can be successfully isolated from different species and cultured for long periods (up to now, the longest culture was maintained for 2 years1,38); typical PE cell morphology, pigmentation and function was observed (Figure 1, .......

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A thank deserves to Gregg Sealy and Alain Conti for their excellent technical assistance. This work was supported by the European Commission in the context of the Seventh Framework Programme, the Swiss National Sciences Foundation, and the Schmieder-Bohrisch Foundation. Z.I. received funding from the European Research Council, ERC Advanced [ERC-2011-ADG 294742] and B.M.W. from a Fulbright Research Grant and Swiss Government Excellence Scholarship.

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Name Company Catalog Number Comments
12-well plates Corning 353043
24-well plates Corning 353047
48-well plates ThermoFisher Scientific 150687
6-well plate Greiner 7657160
Betadine Mundipharma
Bonn micro forceps flat
Colibri forceps (sterile)
CytoTox-Glo Cytotoxicity Assay Promega G9291
DMEM/Ham`s F12 Sigma-Aldrich D8062
Drape (sterile) Mölnlycke Health Care 800530
Electroporation buffer 3P.14 3P Pharmaceutical
FBS Brunschwig P40-37500
Forceps (different size) (sterile)
Gauze compress PROMEDICAL AG 25403
NaCl (0.9%) Laboratorium Dr. Bichsel AG 1000090
Needle (18G)  Terumo TER-NN1838R
Neon Transfection kit 10 µL ThermoFisher Scientific MPK1096
Neon Transfection System ThermoFisher Scientific MPK5000S
Neubauer chamber Marienfeld-superior 640010
Pasteur pipette (fire-polish) Witeg 4100150
PBS 1X Sigma-Aldrich D8537
Penicillin/Streptomycin Sigma-Aldrich P0781-100
Pentobarbital (Thiopental Inresa) Ospedalia AG 31408025
Petri dish ThermoFisher Scientific 150288
pFAR4-PEDF
pFAR4-SB100X
pFAR4-Venus Pastor et al., 2018. Kindly provided by Prof. Scherman and Prof. Marie
pSB100X (250 ng/µL) Mátés et al., 2009. Provide by Prof. Izsvak
pT2-CAGGS-Venus Johnen et al., 2012
pT2-CMV-GMCSF-His plasmid DNA (250 ng/µL) Cloned in our lab
pT2-CMV-PEDF-His plasmid DNA (250 ng/µL) Pastor et al., 2018
scarpel no. 10 Swann-Morton 501
scarpel no. 11 Swann-Morton 503
Sharp-sharp tip curved Extra Fine Bonn Scissors (sterile) 
Sharp-sharp tip straight Extra Fine Bonn Scissors (sterile)
Tali Image-Based Cytometer ThermoFisher Scientific T10796
Trypsin 0.25%  ThermoFisher Scientific 25050014
Trypsin 5%/EDTA 2% Sigma-Aldrich T4174
Vannas spring scissors curved (sterile)

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