Name: isolation, culture, and genetic engineering of mammalian primary pigment epithelial cells for non-viral gene therapy
Uploaded: 2021-02-26T13:30:00
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 (...

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 ...

The protocol is significant since it shows the isolation and transfection of pigment epithelial cells from five mammals as an optimal system to study ocular gene therapy in various setups. The main advantage of this method is its flexibility, transferability, high cell yield, viability and transfection rate due to the five species and the non-viral SB100X transposon system used. These techniques have been developed to study gene therapy approaches targeting retinal and iris pigment epithelial cells, which are isolated and transfected in the present protocol, but are usable for any ocular research.Demonstrating the procedure will be Gregg Sealy, a technician from the laboratory. After euthanizing the animal, use curved scissors and Colibri forceps to enucleate the eyes, then clean the remaining muscle tissue and skin from the eyes. Collect the eyes in a 50 milliliter tube filled with non-sterile PBS and transfer the tube to the laminar flow hood.Disinfect them by submerging in iodine-based solution for two minutes, then transfer the eyes to a 50 milliliter tube filled with sterile PBS. Put one eye on a sterile gauze compress and firmly hold the eye close to the optic nerve. Then cut near the limit of the iris with a number 11 scalpel.Using scissors, cut around the iris and remove the anterior segment and put it in a Petri dish, leaving the bulb with the vitreous until the retinal pigment epithelial, or RPE cells, are isolated. To isolate iris pigment epithelial, or IPE cells, remove the lens with fine forceps and delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish and wash it with sterile PBS.Add 500 microliters of 0.25%trypsin and incubate for 10 minutes at 37 degrees Celsius. Remove the trypsin, add 500 microliters of complete medium, and scrape the IPE delicately with a flat, fire-polished Pasteur pipette. Collect the cell suspension and put it in a 1.5 milliliter tube.Count the cells using the Neubauer chamber, and seed 0.2 million cells per well in a 24 well plate with one milliliter of complete medium. Place the plate in an incubator and culture it at 37 degrees Celsius and 5%carbon dioxide. To isolate RPE cells, remove the vitreous humor and retina from the posterior segment with thin forceps.Put the bulbs in the 24 well plate and add 500 microliters of 0.25%trypsin per eye and incubate for 10 minutes at 37 degrees Celsius. Remove trypsin, add 500 microliters of complete medium per globe and scrape the RPE cells delicately with a curved, fire-polished Pasteur pipette. Collect the cell suspension and put it in a 1.5 milliliter tube.Count the cells using the Neubauer chamber and seed the cells as described previously. Place the plate in the incubator. Clean remaining muscle tissue and skin from the eyes and disinfect the eyes in iodine-based solution and rinse them with PBS.To isolate IPE cells, remove the lens and pull out the iris as described previously. After preparing two irises, add one milliliter of complete medium and isolate the cells by scratching with a flat, fire-polished Pasteur pipette, then count and seed the cells. Place the plate in the incubator.To isolate RPE cells, remove the vitreous and retina from the posterior segment and place the bulb in a Petri dish. Fill the bulb with one milliliter complete medium. Using a curved, fire-polished Pasteur pipette, remove the RPE cells.Collect the cell suspension within the bulb using a 1, 000 microliter pipette, and transfer it into a 1.5 milliliter tube for resuspension. Then count and seed the cells and incubate the plate as previously demonstrated. After cleaning, open the eye and cut around the iris to remove the anterior segment, then put in a Petri dish.Leave the bulb with the vitreous until RPE cells are isolated. To isolate IPE cells, if the lens come with the anterior segment, remove the lens and use fine forceps to delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish and wash it with sterile PBS.Cut the ciliary body with a scalpel number 10. After preparing the irises, incubate them with two milliliters of 0.25%trypsin at 37 degrees Celsius for 10 minutes. Remove the trypsin and add two milliliters of complete medium, then isolate the cells by scratching with a flat, fire-polished Pasteur pipette.Transfer the cell suspension into a two milliliter tube and centrifuge the cells for 10 minutes at 120 times G.Discard the supernatant and resuspend the cells in two milliliters of complete medium. Seed 0.32 million cells per well in a six well plate in three milliliters of complete medium. Place the plate in an incubator and culture it at 37 degrees Celsius and 5%carbon dioxide.To isolate RPE cells, remove the vitreous humor and retina from the posterior segment with forceps. Avoid damaging the retinal pigment epithelium. Wash the bulb with PBS.After preparing both eyes, fill the bulb about about three-fourths full with trypsin, and incubate for 25 minutes at 37 degrees Celsius with the Petri dish lid on top of the bulb eye. Remove the trypsin and add one milliliter of complete medium. Using a curved, fire-polished Pasteur pipette, remove the RPE cells.Collect the cell suspension within the bulb and transfer it into a 1.5 milliliter tube for resuspension. Centrifuge the cells for 10 minutes at 120 times G.Seed 0.32 million cells per well in a six well plate in three milliliters of complete medium. Place the plate in an incubator and culture it at 37 degrees Celsius and 5%carbon dioxide.Culture the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator. After three to four days, pipette up and down to collect nonadherent cells and put half of the volume in another well. Fill the well to one milliliter with complete medium.After another three to four days, repeat the cell collection, this time pooling the nonadherent cells from two wells into one. Add medium to all wells. Observe the cells and change the medium two times per week.When cells reach confluence, switch to complete medium with 1%FBS. Using the aforementioned protocols, IPE and RPE cells were successfully isolated and cultured from five different species. The RPE expression pattern was confirmed in primary bovine IPE cells by immunofluorescence for RPE65.Cell viability was studied to exclude a potential toxicity of the electroporation buffer using a commercially available cytotoxicity assay kit and a knot electroporated control. No impact on cell viability was observed. Precultured pigment epithelial cells from different species were transfected with the yellow fluorescent Venus protein.Transfected RPE19 cells were used as a positive control. The quantification of the transfection efficiency in precultured pig RPE is shown here. Freshly isolated rabbit pigment epithelial cells were transfected with the yellow fluorescent protein Venus and were monitored by microscopy.Pigment epithelial cells were transfected with pigment epithelium derived factor and protein secretion was monitored by Western blot. The signal for transfected cells was higher compared with non-transfected cells for all species and days studied, and no decrease in protein secretion was observed within this time. It is crucial to avoid scraping the cells with sharp tools.Here, fire-polished Pasteur pipettes are used, except in small mouse eyes, where a round scalpel must be used. Pigment epithelial cells can be used to develop gene therapy approaches and to simulate pathological conditions, for example, oxidative stress damage. These models can also be used to test therapeutic drugs.

Research

JoVE Journal

Biology

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 ...

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 ...

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, ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

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 ...

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 ...

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 ...

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 ...

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 ...

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, ...