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

Summary

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.

Abstract

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.

Introduction

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 primary cells (e.g., they are less pigmented) and is, therefore, only of limited value for pre-clinical analyses¹. Additionally, non-human mammalian cells can be purchased for less cost and in bigger quantities; human donor tissue can be obtained from various Eye Banks, but the availability is limited and expensive. Finally, new Advanced Therapy Medicinal Products (ATMP, i.e., cell, tissue, or Gene Therapy Medicinal Product) need to be applied in at least two different species before being tested in patients and these in vivo studies request the preparation of allogenic cell transplants.

Retinal neurodegenerative diseases are the leading cause of blindness in industrialized countries, comprising common diseases like AMD, as well as rare diseases like retinitis pigmentosa, in which the retinal cell death eventually leads to blindness. RPE cells, photoreceptor, and retinal ganglion cells (RGC) damage can in some cases be slowed, but there are currently no curative therapies available. ATMPs offer the potential to correct gene defects, integrate therapeutic genes or replace degenerated cells, thereby enabling the development of regenerative and curative therapies for diseases such as AMD; 13 gene therapies already got marketing approval including a therapy to treat RPE65 mutation-associated retinal degeneration^2,3. Among older adults (>60 years), ~30 million people worldwide are affected by either neovascular (nvAMD) or avascular (aAMD) AMD⁴. Both forms are induced by age-associated triggers including oxidative damage, function impairment and loss of RPE cells followed by photoreceptor degradation, among others (e.g. genetic risk alleles, smoking, hypertension)^5,6. In nvAMD, pathogenesis is aggravated by an imbalance of angiogenic and anti-angiogenic factors in favor of the angiogenic Vascular Endothelial Growth Factor (VEGF) that induces choroidal neovascularization (CNV). To date, only nvAMD is treatable by monthly intravitreal injections of inhibitors of the VEGF protein to suppress the CNV; no effective treatment is yet available for aAMD^6,7.

Several studies evaluated cell-based therapies to replace the anti-VEGF therapy: studies made by Binder et al., in which freshly-harvested autologous RPE cells were transplanted into patients with nAMD^8,9,10, showed moderate visual improvement, but only a small group of patients reached a final visual acuity high enough to enable reading. Recently, a phase I clinical study used an embryonic stem cell-derived RPE patch to treat AMD with promising results; i.e., efficacy, stability, and safety of the RPE patch for up to 12 months in 2 of the 10 patients treated¹¹. In addition, several groups have published studies in which autologous RPE-Bruch's membrane-choroid patches were harvested from the peripheral retina and transplanted to the macula^12,13,14; and induced pluripotent stem cell (iPSC)-derived RPE patches were generated for transplantation¹⁵. For aAMD, antibodies targeting the complement pathway have been tested in clinical trials^6,16 and a phase I study using a single intravitreal injection of an adeno-associated viral (AAV) vector coding the gene for the factor CD59 (AAVCAGsCD59) in patients with geographic atrophy (GA) was completed¹⁷; the phase II study recently started and aims to recruit 132 patients with advanced aAMD and to evaluate the outcome at 2 years post-intervention¹⁸. Finally, the FocuS study group has started a phase I/II multicenter clinical trial evaluating the safety, dose response and efficacy of a recombinant non-replicating AAV vector encoding a human complement factor¹⁹.

Primarily, the goal of a regenerative AMD therapy is the transplantation of functional RPE cells, which were damaged or lost. However, IPE and RPE cells share many functional and genetic similarities (e.g., phagocytosis and retinol metabolism), and because IPE cells are more feasibly harvested, they have been proposed as an RPE substitute²⁰. Even though it has been previously demonstrated that the IPE cell transplantation delays photoreceptor degeneration in animal models^21,22 and stabilizes visual function in patients with end-stage nvAMD, no significant improvement was observed in these patients²³. The lack of efficacy may be due to the low number of transplanted cells, and/or the imbalance of neuroprotective retinal factors. An alternative approach would be to transplant transfected pigment epithelial cells that overexpress neuroprotective factors to restore retinal homeostasis, maintain remaining RPE cells, and protect photoreceptors and RGCs from degeneration. Consequently, we propose a new therapy that comprises the transplantation of functional RPE or IPE cells that have undergone genetic engineering to secrete neuroprotective and anti-angiogenic proteins, such as PEDF, GM-CSF or insulin-like growth factors (IGFs). The advantage of developing and analyzing this approach in several species instead of using a cell line, only one species, or human tissue is: 1) increased reproducibility and transferability of the results as shown by numerous studies realized in independent laboratories and different species^1,24,25; 2) pig or bovine cells are feasibly disposable without the sacrifice of additional animals; 3) the availability of especially pig and bovine cells allows large test series to produce robust results; 4) the knowledge to isolate, culture and genetically modify cells from the mostly used models enables in vivo analyses in multiple species^24,25,26 and thus offers an improved risk-benefit ratio for the first treated patients; 5) the flexibility of the protocol presented allows its use in various models and experimental set ups and for all ocular cell based therapies with and without genetic engineering. In contrast, alternative techniques as cell lines or human tissue are only of limited transferability and/or limited disposability. Cell lines such as the ARPE-19 are ideal for preliminary experiments; however, low pigmentation and high proliferation differ significantly from primary cells¹. RPE and IPE cells, which are isolated from human donor tissue offer a precious source for transferable in vitro experiments; however, we obtain human tissue from an US-American Eye Bank meaning that the tissue is at least two days old (after enucleation) and requires a long and expensive transport, but local donor tissue is not available in sufficient amounts for a productive research. The advantage of the use of primary cells is confirmed by multiple studies from other groups^27,28.

For the development of a cell-based non-viral gene therapy using the SB100X transposon system for transfecting primary RPE and IPE cells with the genes coding for PEDF and/or GM-CSF to treat nvAMD and aAMD, respectively^29,30,31,32, we first established the transfection of ARPE-19 cells¹. Next, the isolation and transfection protocols were established in readily accessible bovine and porcine primary cells. Now, the isolation and transfection of primary RPE and IPE cells from five different species has been established, from small (as mouse) to large mammals (as cattle). It was confirmed in primary RPE and IPE cells derived from human donor eyes³⁰. The Good Manufacturing Practices (GMP)-compliant production of the ATMP was validated using human donor tissue as well³³. Finally, both safety and efficiency of the approach were assessed in vivo in three different species for which the protocol has been adapted: mouse, rat, and rabbit. In the clinical set-up, an iris biopsy will be harvested from the patient and IPE cells will be isolated and transfected in the clean room, before the cells will be transplanted subretinally back into the same patient. The entire process will take place during a single surgical session that lasts approximately 60 minutes. The development of the treatment approach and the evaluation of its efficiency requested excellent in vitro and ex vivo models to implement robust and efficient gene delivery methods, to analyze efficiency of gene delivery, therapeutic protein production and neuroprotective effects, and to produce cell transplants to test the approach in vivo^{1,24,25,29,30}. It is worth mentioning that the therapy has the ethical approval for a clinical phase Ib/IIa trial from the ethical commission for research of the Canton of Geneva (no. 2019-00250) and currently last pre-clinical data requested for authorization by Swiss regulatory authorities are collected using the presented protocol. In this regard, pre-clinical in vivo data demonstrated a significant reduction in CNV and excellent safety^24,25,31.

Here, the isolation and culture of RPE/IPE cells from bovine, pig, rabbit, rat and mouse, and the use of the integrative SB100X transposon system combined with electroporation as an efficient gene-delivery method is described. Particularly, primary PE cells were transfected to overexpress PEDF and GM-CSF. The collection of these protocols enables the in vitro and in vivo studies to be performed in all pre-clinical phases of ATMP development. Moreover, the set-up has the potential to be adapted to other genes of interest and diseases.

Protocol

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 (150 mg/kg) diluted in 0.9% NaCl injected intraperitoneal and the eyes were enucleated immediately after sacrifice. Porcine and bovine eyes were obtained from a local slaughterhouse within 6 hours of sacrifice and were transported to the laboratory on ice.

1. Before preparation

1. Prepare complete medium (DMEM/Ham's F12 supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin / 80 µg/mL streptomycin, and 2.5 µg/mL amphotericin B). Heat the medium, 1x PBS, and 0.25% trypsin (if necessary) in a 37 °C water bath.
2. Put a sterile drape into the hood to prepare an aseptic working place. Introduce all needed sterile instruments and materials inside the hood.
   ​NOTE: Only the enucleation of the eyes and cleaning of remaining muscle tissue and skin are the procedures carried out outside the hood, the rest of the steps must be performed inside the hood.

2. Isolation of rat/mouse PE cells

1. Use curved scissors and Colibri forceps to enucleate the eyes after euthanizing the animal. Clean the remaining muscle tissue and skin from the eyes using scissors and forceps (non-sterile).
   ​NOTE: The size of the scissors and forceps used for the enucleation and cleaning of the eyes depends on the species (e.g., for rat and mouse the instruments are going to be smaller than the ones used for pig and cattle) (see Figure S1).
   1. Collect the eyes in a 50 mL tube filled with non-sterile PBS and transfer the tube to the laminar flow hood. Disinfect the eyes by submerging for 2 min in iodine-based solution, then transfer them to a Petri dish filled with sterile PBS.
2. Opening of the bulb
   1. After transferring the eyes to a sterile Petri dish, hold one firmly close to the optic nerve with Colibri or pointed forceps. Punch a hole near the limit of the iris (between pars plana and ora serrata) with an 18 G needle. Insert small scissors in the hole and cut around the iris. Remove the anterior segment (cornea, lens, and iris) and put it in a Petri dish. Leave the bulb with the vitreous until RPE cells are isolated.
3. Isolation of IPE cells
   1. Remove the lens and with fine forceps delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish, wash with sterile PBS and leave it in PBS until more iris are prepared.
   2. Repeat steps 2.2.1 to 2.3.1 for all eyes to be prepared that day.
   3. Add 50 µL of 0.25% trypsin per iris and incubate for 10 min at 37 °C. Remove trypsin, add 150 µL of complete medium per iris and scrape the IPE delicately with a flat fire-polished Pasteur pipette; use fine forceps to immobilize the tissue. Collect the cell suspension and put in a 1.5 mL tube. Use 10 µL of the cell suspension diluted 1:3 with trypan blue to count the cells in the Neubauer chamber^34,35.
   4. If not transfected immediately, seed 200,000 cells/well in a 24-well plate (100,000 cells/cm²) in 1 mL of complete medium (10% FBS) (for seeding see Table 1). Place the plate in an incubator and culture it at 37 °C, 5% CO₂.
      NOTE: It might be necessary to pool several eyes together to have enough cells for seeding.
4. Isolation of RPE cells
   1. Remove vitreous humor and retina from the posterior segment with thin forceps. Avoid damaging the retinal pigment epithelium.
   2. Cut the segment in half with a #10 scalpel to have the globe completely open and wash with sterile PBS.
   3. Add 50 µL of 0.25% trypsin per eye and incubate for 10 min at 37 °C. Remove trypsin and add 150 µL of complete medium per globe and scrape the RPE cells delicately with a round scalpel; use fine forceps to immobilize the tissue. Collect the cell suspension and put it in a 1.5 mL tube. Take 10 µL of the cell suspension; dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.
   4. See step 2.3.4.
      ​NOTE: It might be necessary to pool several eyes together to have enough cells for seeding.

3. Isolation of rabbit PE cells

1. Perform cleaning and disinfection as described in steps 2.1 to 2.1.1.
2. Put one eye on a sterile gauze compress and hold it firmly close to the optic nerve. Open the eye with the scalpel #11 and scissors about 2 mm under the limbus. Remove the anterior segment (cornea, lens, and iris) and put it in a Petri dish. Leave the bulb with the vitreous until RPE cells are isolated.
3. Isolation of IPE cells
   1. Perform step 2.3.1. Remove the ciliary body from the iris by cutting with a scalpel #10.
   2. After the preparation of 2 iris, incubate with 1 mL of 0.25 % trypsin at 37 °C for 10 min; during this time, the RPE cells can be isolated (see step 3.4). Remove the trypsin and add 1 mL complete medium to the iris and isolate the cells by carefully scratching with a flat fire-polished Pasteur pipette. Resuspend the cells carefully by pipetting and transfer the cell suspension into a 1.5 mL tube. Take 10 µL of the cell suspension and dilute 1:3 with trypan blue to count the cells in the Neubauer chamber.
   3. See step 2.3.4.
4. Isolation of RPE cells
   1. Perform step 2.4.1.
   2. Put a sterile gauze into a 12 well plate and put the bulb over the gauze.
   3. Wash with PBS and perform step 2.4.3.
      NOTE: Use a curved fire-polished Pasteur pipette.
   4. Centrifuge the cells 10 min at 120 x g.
   5. See step 2.3.4.

4. Isolation of pig PE cells

1. Perform cleaning as described in step 2.1. Wash with PBS and disinfect the eyes by submerging for 2 min in iodine-based solution, rinse with PBS. Continue with step 3.2.
2. Isolation of IPE cells
   1. Perform step 3.3.1. After the preparation of 2 iris, add 1 mL of complete medium and isolate the cells by carefully scratching with a flat fire-polished Pasteur pipette. Transfer the cell suspension into a 1.5 mL tube. Take 10 µL of the cell suspension and dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.
   2. See step 2.3.4.
3. Isolation of RPE cells
   1. Perform step 2.4.1. Place the bulb in a Petri dish and wash with PBS. Fill the bulb with 1 mL complete medium.
   2. Using a curved fire-polished Pasteur pipette, carefully remove RPE cells. Make sure to scrape from the bottom to the top to avoid slipping down of the choroid-Bruch's membrane complex. Collect the cell suspension within the bulb using a 1,000 µL pipette and transfer into a 1.5 mL tube for resuspension. Take 10 µL of the cell suspension and dilute 1:8 with trypan blue to count the cells in the Neubauer chamber.
   3. See step 2.3.4.

5. Isolation of bovine PE cells

1. Perform cleaning as described in step 2.1. Wash with PBS and disinfect the eyes by submerging for 2 min in iodine-based solution, rinse with PBS. Continue with step 3.2.
2. Isolation of IPE cells
   1. Perform step 3.3.1. After the preparation of two iris, incubate with 2 mL of 0.25% trypsin at 37 °C for 10 min. During this time, the RPE cells can be isolated (see step 5.3).
   2. Remove the trypsin and add 2 mL complete medium to the iris and isolate the cells by carefully scratching with a flat fire-polished Pasteur pipette. Transfer the cell suspension into a 15 mL tube. Centrifuge the cells 10 min at 120 x g. Take 10 µL of the cell suspension and dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.
   3. If not transfected immediately, seed 320,000 cells/well in a 6-well plate in 3 mL of complete medium (10% FBS) (for seeding see Table 1). Place the plate in an incubator and culture it at 37 °C, 5% CO₂.
3. Isolation of RPE cells
   1. Follow step 2.4.1. Place the bulb in a Petri dish and wash with PBS. After preparation of 2 eyes fill the bulb about ¾ with trypsin and incubate for 25 min at 37 °C with the lid of the Petri dish on top of the bulbi.
   2. Remove the trypsin and add 1 mL complete medium. Perform step 4.3.2. Centrifuge the cells 10 min at 120 x g.
   3. See step 5.2.3.

6. Cultivation - medium change

1. Culture the cells in DMEM/Ham's F12, supplemented with 10% FBS, 80 U/mL penicillin / 80 µg/mL streptomycin, and 2.5 µg/mL amphotericin B, at 37°C and 5% CO₂ in a humidified incubator. After 3-4 days pipette up and down to collect non-adherent cells and put half of the volume in another well. Fill up to 1 mL with complete medium.
   NOTE: This allows having a surface large enough for all cells isolated to attach and maximize the output.
2. After another 3-4 days repeat the cell collection but this time by pooling the non-adherent cells from two wells into one well (e.g., A1+B1 in C1). Add medium to all wells. Observe the cells and change the medium 2 times per week (for 6 and 24-well plates use 3 and 1 mL/well, respectively). When cells reach confluence, switch to complete medium with 1% FBS or use the cells for experiments (e.g., transfection).
   ​NOTE: RPE and IPE cells are confluent after 3-4, and 4-5 weeks after isolation, respectively. The cell culture purity was corroborated checking the cell morphology (pigmented cells) and specific markers as described by Johnen and colleagues³⁶.

7. Electroporation of primary PE cells

1. Perform electroporation as described before^1,37.
2. Depending on the number of cells transfected use 6-, 24- or 48-well plates (see Table 2) to seed the cells in medium without antibiotics or antimycotics. For the following 2 weeks add drops with medium containing penicillin (80 U/mL), streptomycin (80 µg/mL), and amphotericin B (2.5 µg/mL) twice a week. Exchange the medium completely 2 weeks after transfection.
3. To determine cell growth, transfection efficiency, and protein secretion, monitor the cells weekly by microscopy and analyze the cell culture supernatant by western blot. Before termination of the culture, take a 24 h cell culture supernatant to quantify protein secretion by ELISA, count the cells, measure fluorescence by image-based cytometry (in case of Venus-transfected cells) following manufactures' instructions (see Table of Materials), and collect the cell pellet for RT-qPCR-based gene expression analysis.
   NOTE: These methods are not included in the present paper since it is not the purpose to explain in detail the analysis of the cells but rather their isolation. The cell seeding density is the same for all species (100,000 cells/cm²) but because the number of isolated cells varies, different plates were used. In addition, for mice, rat, and rabbit it might be necessary to pool 2-3 eyes to have enough cells for seeding.

Species	Trypsin treatment	N° IPE cells	N° RPE cells	Plate for seeding (100,000 cells/cm2)
Mouse/rat	Yes	~50,000	~150,000	24-well plates
Rabbit	Yes	~350,000	~2,500,000	24-well plates
Pig	No	~1,000,000	~3,000,000	24-well plates
Bovine	Yes	~1,700,000	~5,000,000	6-well plates

Table 1: Number of primary PE cells isolated from eyes from different species.

Name	Area	Volume medium	Trypsin	Volume medium to stop action of trypsin	Seeding density
6-well plate	9.6 cm²	3.0 mL	0.5 mL	1.0 mL	3x105
24-well plate	2.0 cm²	1.0 mL	0.2 mL	0.8 mL	5x104
48-well plate	1.1 cm²	0.5 mL	0.1 mL	0.4 mL	0.5-1x104

Table 2: Cell culture volumes and seeding densities.

Results

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-isolation,...

Discussion

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 years^1,38); typical PE cell morphology, pigmentation and function was observed (Figure 1, ...

Materials

List of materials used in this article

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)