Name: isolation, culture, and genetic engineering of mammalian primary pigment epithelial cells for non-viral gene therapy
Uploaded: 2021-02-26T13:30:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy at JoVE.com

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.

The protocols in which animals were involved were carried out by certified personnel and after authorization by the cantonal D&#233;partement de la s&#233;curit&#233;, de l&#39;emploi et de la sant&#233; (DSES), Domaine de l&#39;exp&#233;rimentation 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
<ol>
	<li>Prepare complete medium (DMEM/Ham&#39;s F12 supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin / 80 &#181;g/mL streptomycin, and 2.5 &#181;g/mL amphotericin B). Heat the medium, 1x PBS, and 0.25% trypsin (if necessary) in a 37 &#176;C water bath.</li>
	<li>Put a sterile drape into the hood to prepare an aseptic working place. Introduce all needed sterile instruments and materials inside the hood. 
	&#8203;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.</li>
</ol>
2. Isolation of rat/mouse PE cells
<ol>
	<li>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). 
	&#8203;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&#160;than the ones used for pig and cattle) (see Figure S1).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Opening of the bulb
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Isolation of IPE cells
	<ol>
		<li>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.</li>
		<li>Repeat steps 2.2.1 to 2.3.1 for all eyes to be prepared that day.</li>
		<li>Add 50 &#181;L of 0.25% trypsin per iris and incubate for 10 min at 37 &#176;C. Remove trypsin, add 150 &#181;L 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 &#181;L of the cell suspension diluted 1:3 with trypan blue to count the cells in the Neubauer chamber34,35.</li>
		<li>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 &#176;C, 5% CO2. 
		NOTE: It might be necessary to pool several eyes together to have enough cells for seeding.</li>
	</ol>
	</li>
	<li>Isolation of RPE cells
	<ol>
		<li>Remove vitreous humor and retina from the posterior segment with thin forceps. Avoid damaging the retinal pigment epithelium.</li>
		<li>Cut the segment in half with a #10 scalpel to have the globe completely open and wash with sterile PBS.</li>
		<li>Add 50 &#181;L of 0.25% trypsin per eye and incubate for 10 min at 37 &#176;C. Remove trypsin and add 150 &#181;L 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 &#181;L of the cell suspension; dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.</li>
		<li>See step 2.3.4. 
		&#8203;NOTE: It might be necessary to pool several eyes together to have enough cells for seeding.</li>
	</ol>
	</li>
</ol>
3. Isolation of rabbit PE cells
<ol>
	<li>Perform cleaning and disinfection as described in steps 2.1 to 2.1.1.</li>
	<li>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.</li>
	<li>Isolation of IPE cells
	<ol>
		<li>Perform step 2.3.1. Remove the ciliary body from the iris by cutting with a scalpel #10.</li>
		<li>After the preparation of 2 iris, incubate with 1 mL of 0.25 % trypsin at 37 &#176;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 &#181;L of the cell suspension and dilute 1:3 with trypan blue to count the cells in the Neubauer chamber.</li>
		<li>See step 2.3.4.</li>
	</ol>
	</li>
	<li>Isolation of RPE cells
	<ol>
		<li>Perform step 2.4.1.</li>
		<li>Put a sterile gauze into a 12 well plate and put the bulb over the gauze.</li>
		<li>Wash with PBS and perform step 2.4.3. 
		NOTE: Use a curved fire-polished Pasteur pipette.</li>
		<li>Centrifuge the cells 10 min at 120 x g.</li>
		<li>See step 2.3.4.</li>
	</ol>
	</li>
</ol>
4. Isolation of pig PE cells
<ol>
	<li>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.</li>
	<li>Isolation of IPE cells
	<ol>
		<li>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 &#181;L of the cell suspension and dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.</li>
		<li>See step 2.3.4.</li>
	</ol>
	</li>
	<li>Isolation of RPE cells
	<ol>
		<li>Perform step 2.4.1. Place the bulb in a Petri dish and wash with PBS. Fill the bulb with 1 mL complete medium.</li>
		<li>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&#39;s membrane complex. Collect the cell suspension within the bulb using a 1,000 &#181;L pipette and transfer into a 1.5 mL tube for resuspension. Take 10 &#181;L of the cell suspension and dilute 1:8 with trypan blue to count the cells in the Neubauer chamber.</li>
		<li>See step 2.3.4.</li>
	</ol>
	</li>
</ol>
5. Isolation of bovine PE cells
<ol>
	<li>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.</li>
	<li>Isolation of IPE cells
	<ol>
		<li>Perform step 3.3.1. After the preparation of two iris, incubate with 2 mL of 0.25% trypsin at 37 &#176;C for 10 min. During this time, the RPE cells can be isolated (see step 5.3).</li>
		<li>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 &#181;L of the cell suspension and dilute 1:4 with trypan blue to count the cells in the Neubauer chamber.</li>
		<li>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 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>Isolation of RPE cells
	<ol>
		<li>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 &#190; with trypsin and incubate for 25 min at 37 &#176;C with the lid of the Petri dish on top of the bulbi.</li>
		<li>Remove the trypsin and add 1 mL complete medium. Perform step 4.3.2. Centrifuge the cells 10 min at 120 x g.</li>
		<li>See step 5.2.3.</li>
	</ol>
	</li>
</ol>
6. Cultivation - medium change
<ol>
	<li>Culture the cells in DMEM/Ham&#39;s F12, supplemented with 10% FBS, 80 U/mL penicillin / 80 &#181;g/mL streptomycin, and 2.5 &#181;g/mL amphotericin B, at 37&#176;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.</li>
	<li>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). 
	&#8203;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.</li>
</ol>
7. Electroporation of primary PE cells
<ol>
	<li>Perform electroporation as described before1,37.</li>
	<li>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 &#181;g/mL), and amphotericin B (2.5 &#181;g/mL) twice a week. Exchange the medium completely 2 weeks after transfection.</li>
	<li>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&#39; 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.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Trypsin treatment</td>
			<td>N&#176; IPE cells</td>
			<td>N&#176; RPE cells</td>
			<td>Plate for seeding (100,000 cells/cm2)</td>
		</tr>
		<tr>
			<td>Mouse/rat</td>
			<td>Yes</td>
			<td>~50,000</td>
			<td>~150,000</td>
			<td>24-well plates</td>
		</tr>
		<tr>
			<td>Rabbit</td>
			<td>Yes</td>
			<td>~350,000</td>
			<td>~2,500,000</td>
			<td>24-well plates</td>
		</tr>
		<tr>
			<td>Pig</td>
			<td>No</td>
			<td>~1,000,000</td>
			<td>~3,000,000</td>
			<td>24-well plates</td>
		</tr>
		<tr>
			<td>Bovine</td>
			<td>Yes</td>
			<td>~1,700,000</td>
			<td>~5,000,000</td>
			<td>6-well plates</td>
		</tr>
	</tbody>
</table>
Table 1: Number of primary PE cells isolated from eyes from different species.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Area</td>
			<td>Volume medium</td>
			<td>Trypsin</td>
			<td>Volume medium to stop action of trypsin</td>
			<td>Seeding density</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>9.6 cm&#178;</td>
			<td>3.0 mL</td>
			<td>0.5 mL</td>
			<td>1.0 mL</td>
			<td>3x105</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>2.0 cm&#178;</td>
			<td>1.0 mL</td>
			<td>0.2 mL</td>
			<td>0.8 mL</td>
			<td>5x104</td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>1.1 cm&#178;</td>
			<td>0.5 mL</td>
			<td>0.1 mL</td>
			<td>0.4 mL</td>
			<td>0.5-1x104</td>
		</tr>
	</tbody>
</table>
Table 2: Cell culture volumes and seeding densities.

The authors have nothing to disclose.

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, ...

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&#160;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 degeneration2,3. Among older adults (&#62;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 aAMD6,7.
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 nAMD8,9,10, 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&#39;s membrane-choroid patches were harvested from the peripheral retina and transplanted to the macula12,13,14; 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 trials6,16 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 models21,22 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 species1,24,25; 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&#160;modify&#160;cells from the mostly used models enables in vivo analyses in multiple species24,25,26 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&#160;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 groups27,28.
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, respectively29,30,31,32, 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 vivo1,24,25,29,30. 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 safety24,25,31.
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.

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

isolation, culture, and genetic engineering of mammalian primary pigment epithelial cells for non-viral gene therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;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&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

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...

Watch this Scientific Journal Video about Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy at JoVE.com

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

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&#160;analyses and&#160;in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

isolation-culture-genetic-engineering-mammalian-primary-pigment

Research

JoVE Journal

Biology

3.1K Views.  University of Geneva. 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.

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

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

Medicine

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Neuroscience

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...

Isolating RPE and IPE Cells from Bovine Eye: A Procedure to  Harvest and Culture Bovine Primary Pigment Epithelial Cells

Source: Bascuas, T. et al., <a href="https://www.jove.com/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
This video demonstrates the procedure to isolate the retinal pigment epithelial, or RPE, cells and iris pigment epithelial, or IPE, cells from harvested bovine eyes. The isolated cells can be cultured to study their role in the treatment of ocular diseases.

Source: Bascuas, T. et al., Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...

JoVE Encyclopedia of Experiments

Large Animal Models

Bovine

Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye

Source: Bascuas, T., et al. <a href="https://www.jove.com/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
In this video, we show a procedure to obtain ocular pigment epithelial cells from a rabbit model. The cells are helpful in developing therapeutic strategies for retinal degenerative diseases.

Source: Bascuas, T., et al. Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...