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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#246;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)&#160;</td>
			<td>Terumo</td>
			<td>TER-NN1838R</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection kit 10 &#181;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/&#181;L)</td>
			<td></td>
			<td></td>
			<td>M&#225;t&#233;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/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Cloned in our lab</td>
		</tr>
		<tr>
			<td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&#181;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)&#160;</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%&#160;</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 ...

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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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...