Name: isolation of primary porcine retinal pigment epithelial cells for in vitro modeling
Uploaded: 2024-05-03T13:20:00.000+00:00
Duration: 6 min 37 s
Description: Watch this Scientific Journal Video about Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models at JoVE.com

The authors would like to thank Farhad Farjood for help with porcine RPE cell culture and isolation and Thomas Harris for help with SEM. The authors acknowledge the support from the Microscopy Core Facility at Utah State University for the SEM analysis. The facility maintains a scanning electron microscope acquired through a National Science Foundation Major Research Instrumentation Grant (CMMI-1337932). Funding for this study was provided by a National Institutes of Health through Grant 1R15EY028732 (Vargis) and a BrightFocus Foundation Grant M2019109 (Vargis). Additional funding was provided by an Undergraduate Research and Creative Opportunities Grant (Weatherston) from Utah State University&#39;s Office of Research and a seed grant (Vargis) from Utah State University&#39;s Alzheimer&#39;s Disease and Dementia Research Center.

The eyes used in this procedure are obtained post-mortem from a local, USDA-inspected butcher shop, and no work is performed using live animals. After the animals have been sacrificed, approximately 2 h pass before the eyes are enucleated. As tissue decay may begin to occur, it is important to keep the eyes cool during transport to prevent further decay. In this procedure, the eyes are immediately placed into a refrigerator after enucleation. Subsequently, the bag of eyes is positioned inside a 1000 mL polypropylene beaker and surrounded by ice within an 8 L cooler. It is important not to place the eyes directly on the ice. Once the eyes arrive at the laboratory, the isolation of RPE cells is carried out within a biosafety cabinet. It is crucial to complete this process within 3-5 h. This protocol has been optimized for processing 10 eyes.
1. Preparing DNase stock aliquots
NOTE: It is recommended that this step is performed before the isolation.
<ol>
	<li>Prepare a 5 mM calcium chloride solution with sterile deionized water. 
	CAUTION: Calcium chloride powder can cause severe eye irritation or damage. Wear appropriate PPE.</li>
	<li>Add 5 mM calcium chloride solution to DNase powder (see Table of Materials) to get a final DNase concentration of 3 mg/mL. Ensure the solution is mixed thoroughly. 
	&#8203;CAUTION: DNase is a respiratory sensitization hazard. Do not inhale powder/substance.</li>
	<li>Aliquot small volumes, for example 1 mL, into microcentrifuge tubes.</li>
	<li>Freeze aliquots at -20 &#176;C until use.</li>
</ol>
2. Setting up the dissection area
<ol>
	<li>Transfer the sterile dissection instruments and cell culture equipment into the biosafety cabinet: surgical drape, Petri dishes, cell strainers, well plates, gauze, tweezers, scalpel handle, scalpel blade, and iris scissors (see Table of Materials).</li>
	<li>Using tweezers, lay out the surgical drape. Place one 6-well plate atop the surgical drape and remove the lid.</li>
	<li>Place an aliquot of sterile Dulbecco&#39;s Phosphate Buffered Saline (DPBS) on ice and into the biosafety cabinet.</li>
	<li>Fill one well of a 6-well plate halfway full (roughly 6 mL) of 10% povidone-iodine solution.</li>
	<li>Fill the remaining wells with chilled DPBS.</li>
	<li>Remove the lid from one of the Petri dishes and place it, inverted, atop the surgical drape. Set aside the Petri dish base as a waste container.</li>
	<li>Place an aliquot of cell culture media (Dulbecco&#39;s Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS), 1% antibiotics/antimycotics) and an aliquot of 0.25% trypsin/EDTA into a 37 &#176;C water bath.</li>
	<li>Remove the DNase stock solution from the freezer (see rest of protocol for more details).</li>
</ol>
3. Exterior tissue removal
NOTE: Exterior tissue removal can be performed outside of the biosafety cabinet. Inspect the eye prior to cutting to ensure that the sclera has not been punctured, the pupil is not foggy, and the optic nerve is present. Discard eyes not meeting these criteria.
<ol>
	<li>While holding the muscle attached to the eye with one hand, use the other hand to gently cut away the tissue surrounding the sclera with a scalpel. Be careful not to crush the eye or accidentally cut through the sclera. If the sclera is cut through and exposes the black interior, discard the eye. 
	CAUTION: A cut resistant glove is highly recommended for the hand holding the muscle to protect against accidental cuts.</li>
	<li>Trim the optic nerve with curved iris scissors to a length of no greater than 3 mm. If the optic nerve is too long, the eye cup will not sit properly in the cell strainer later.</li>
	<li>Place the eye onto ice with the cornea down.</li>
	<li>Repeat steps 3.1-3.3 until the exterior tissue is removed from each eye.</li>
</ol>
4. Eye dissection
<ol>
	<li>Use tweezers to transfer an eye into the biosafety cabinet and into the well containing 10% povidone-iodine solution and rotate the eye to ensure that it is fully coated in the iodine solution. To keep the tools as sterile as possible, these tweezers should only be used for touching the exterior of the eye.</li>
	<li>While the eye is sitting in iodine solution, place a sterile gauze onto the shallow, inverted lid of the Petri dish prepared earlier. In a separate Petri dish, place a cell strainer. Only use the cell strainer to support the eye cup, not for cell separation.</li>
	<li>After allowing the eye to sit in iodine solution for roughly 30 s, wash the eye by dipping it into the wells containing DPBS for roughly 5 s each, moving from well to well to progressively dilute the iodine solution, and transfer the eye onto the sterile gauze.</li>
	<li>Make a small incision using a scalpel below the ora serrata, or roughly 1/3 down the globe from the iris edge.</li>
	<li>Following the incision, use iris scissors to cut evenly along the circumference of the globe, parallel to the cornea. When maneuvering the eye, only use the tweezers to touch the outside of the eye or the gauze to maintain sterility.</li>
	<li>Use the tweezers to grab the optic nerve and gently lift the globe away from the gauze. The vitreous should fall out of the globe. If needed, gently shake the eye to help dislodge the vitreous. 
	NOTE: If the vitreous is very difficult to remove, try cutting lower on the globe.</li>
	<li>Gently place the eye cup into the prepared cell strainer, with the interior of the eye cup facing up. If uneven, use tweezers to adjust the eye cup to get the incision as level as possible.</li>
	<li>Gently add cooled DPBS to the eye cup so the fluid level is just below the lowest point of the incision. This DPBS will be removed following neural retina removal. 
	NOTE: The volume of DPBS added will vary depending on eye size and incision location.</li>
	<li>Repeat steps 4.1-4.8 until all eyes are dissected.</li>
</ol>
5. Neural retina removal and RPE dissociation
NOTE: The following section is easier to perform in stages. In this set up, this process is performed on one batch (usually 3 eye cups) at a time. For steps 5.1-5.7, each step should be performed on the entire batch before moving to the next step.
<ol>
	<li>Under a flashlight, find any areas where the neural retina (white/pink) is starting to detach from the RPE (black). Then, use blunt tweezers to gently grab the lifted neural retina and peel it away. 
	NOTE: If there are no areas where the neural retina is detaching, use the tweezers to gently grab it near the top of the eye cup. Using this method, if damage is done to the RPE/choroid, those cells will not be dissociated during RPE collection.</li>
	<li>Gently collect the neural retina near the optic disc.</li>
	<li>Aspirate out the neural retina using a Pasteur pipette attached to a vacuum aspiration system. It is recommended to feather the aspiration to help in the removal process. 
	NOTE: A pipette tip can be placed on the end of a Pasteur pipette to reduce suction.</li>
	<li>Carefully add additional cooled DPBS to replace what was aspirated.</li>
	<li>Remove remaining neural retina by aspirating once more. This time, remove as much DPBS as possible while not disturbing the exposed RPE.</li>
	<li>Gently add trypsin to the eye cup. Keep the volume level below the incision line and any sections of damaged RPE to reduce contamination. 
	CAUTION: Trypsin is an enzyme that will cause skin and eye irritation on contact. Wear appropriate PPE.</li>
	<li>After trypsin is added to each eye, ensure that the Petri dish lid is replaced. Next, transfer the Petri dish to a humidified incubator at 37 &#176;C and 5% CO2 for 30 min.</li>
	<li>Repeat steps 5.1-5.7 for each batch until all eye cups have been processed.</li>
</ol>
6. RPE collection
<ol>
	<li>Collect the RPE cells from each batch of eyes (2-3 eyes) and place in a single 15 mL centrifuge tube to make the seeding process straightforward.
	<ol>
		<li>Under a flashlight, gently triturate using a 1000 &#181;L pipette to dislodge the RPE cells. If the RPE cells are not detaching, add fresh trypsin and incubate for an additional 10-15 min. Be careful not to scrape the retina.</li>
		<li>Collect the RPE cells in a 15 mL centrifuge tube.</li>
		<li>Wash the eye cup with cell culture media to collect any remaining cells and neutralize the trypsin. Ensure there is at least twice the volume of media to trypsin in the 15 mL centrifuge tube.</li>
	</ol>
	</li>
	<li>Repeat step 6.1 until RPE cells have been collected from all eye cups.</li>
	<li>Centrifuge the cell suspensions at 250 x g for 5 min at room temperature.</li>
</ol>
7. Preparing DNase working solution
<ol>
	<li>Calculate the volume of 3 mg/mL DNase stock solution (prepared in step 1) needed to add to cell culture media for a final concentration of 100 &#181;g/mL and final volume of 3 mL per tube of cells (i.e., for three centrifuge tubes of cells, 9 mL of 100 &#181;g/mL DNase solution is required).</li>
	<li>Combine the appropriate volumes of DNase stock solution and cell culture media. Using DNase in the working solution helps prevent cell clumping and allows cells to be seeded evenly.</li>
</ol>
8. RPE cell seeding
<ol>
	<li>Following centrifugation in step 6.3, remove supernatants from each 15 mL centrifuge tube, careful not to disrupt the cell pellet.</li>
	<li>Resuspend each cell pellet in 3 mL of DNase working solution.</li>
	<li>Place the 15 mL centrifuge tubes containing the resuspended cells into a humidified incubator at 37 &#176;C and 5% CO2 for 15 min.</li>
	<li>Following incubation, centrifuge the cell suspensions at 150 x g for 5 min at room temperature.</li>
	<li>Remove the supernatants, careful not to disrupt the cell pellets.</li>
	<li>Resuspend each cell pellet in 3 mL of fresh cell culture media.</li>
	<li>For each centrifuge tube containing 2-3 eyes worth of RPE cells, seed one well of a 6-well plate (passage 0).</li>
	<li>Carefully, transfer the seeded 6-well plate into a humidified incubator at 37 &#176;C and 5% CO2.</li>
</ol>
9. Primary porcine RPE cell culture
<ol>
	<li>Allow the newly seeded RPE cells to attach for 48-72 h before moving the plate. During the first media change, a wash with fresh cell culture media can be performed to help remove excess cell debris.</li>
	<li>Replace the cell culture media every 48 h.</li>
	<li>When cells reach confluency, transition to a cell culture media with an FBS concentration of 2% and maintain until experimentation.</li>
</ol>
10. RPE cell validation
<ol>
	<li>Immunocytochemical (ICC) staining
	<ol>
		<li>Fix cells in 4% formaldehyde in DPBS for 10 min at room temperature.</li>
		<li>Wash twice with DPBS.</li>
		<li>Permeabilize cells (if needed) using 0.2% Triton-X 100 in DPBS for 10 min at room temperature.</li>
		<li>Wash twice with DPBS.</li>
		<li>Apply blocking solution of 3% (w/v) bovine serum albumin (BSA) in DPBS for 1 h at room temperature.</li>
		<li>Add primary antibody at 1:100 dilution (or recommended manufacturer concentration) (see Table of Materials) and let sit for 1 h at room temperature or overnight at 4 &#176;C.</li>
		<li>Wash three times with DPBS for 5 min each.</li>
		<li>Add secondary antibody (if needed) at 2 &#181;g/mL (or recommended manufacturer concentration) (see Table of Materials) and let sit for 1 h at room temperature.</li>
		<li>Wash three times with DPBS for 5 min each if secondary antibody is applied.</li>
		<li>Add nuclear staining probe at manufacturer&#39;s concentration (see Table of Materials) and let sit for 25 min at room temperature.</li>
		<li>Wash three times with PBS for 5 min each.</li>
		<li>If on a cell culture insert, mount to microscope slide with a coverslip using mounting medium as outlined by Rickabaugh and Weatherston et al.20. Otherwise, image cells in DPBS.</li>
	</ol>
	</li>
	<li>Scanning electron microscopy (SEM)
	<ol>
		<li>Follow the procedure outlined by Harris et al.21.</li>
	</ol>
	</li>
	<li>Trans Epithelial Electrical Resistance (TEER)
	<ol>
		<li>Follow manufacturer&#39;s protocol for TEER measurement (see Table of Materials) with 1 mL of culture medium in the basal chamber15.</li>
	</ol>
	</li>
	<li>Enzyme-linked immunosorbent assay (ELISA)
	<ol>
		<li>Perform ELISA15 using a multiplex human enzyme-linked immunosorbent assay kit following the manufacturer&#39;s protocol (see Table of Materials).</li>
	</ol>
	</li>
</ol>

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

<ol>
	<li>Booij, J. C., Baas, D. C., Beisekeeva, J., Gorgels, T. G. M. F., Bergen, A. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+nature+of+Bruch's+membrane.">The dynamic nature of Bruch's membrane.</a> Progress in Retinal and Eye Research. 29 (1), 1-18 (2010).</li><li>Caceres, P. S., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelium+polarity+in+health+and+blinding+diseases.">Retinal pigment epithelium polarity in health and blinding diseases.</a> Current Opinion in Cell Biology. 62, 37-45 (2020).</li><li>Georgiadis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tight+junction+associated+signalling+proteins+ZO-1+and+ZONAB+regulate+retinal+pigment+epithelium+homeostasis+in+mice.">The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice.</a> PLOS One. 5 (12), e15730 (2010).</li><li>Sparrow, J. R., Hicks, D., Hamel, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+health+and+disease.">The retinal pigment epithelium in health and disease.</a> Current Molecular Medicine. 10 (9), 802-823 (2010).</li><li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiological Reviews. 85 (3), 845-881 (2005).</li><li>Yang, S., Zhou, J., Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+diseases+of+the+retinal+pigment+epithelium.">Functions and diseases of the retinal pigment epithelium.</a> Frontiers in Pharmacology. 12, 727870 (2021).</li><li>Moiseyev, G., Chen, Y., Takahashi, Y., Wu, B. X., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE65+is+the+isomerohydrolase+in+the+retinoid+visual+cycle.">RPE65 is the isomerohydrolase in the retinoid visual cycle.</a> Proceedings of the National Academy of Sciences. 102 (35), 12413-12418 (2005).</li><li>Uppal, S., Liu, T., Poliakov, E., Gentleman, S., Redmond, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+roles+of+RPE65+S-palmitoylation+in+membrane+association+and+visual+cycle+function.">The dual roles of RPE65 S-palmitoylation in membrane association and visual cycle function.</a> Scientific Reports. 9 (1), 5218 (2019).</li><li>Somasundaran, S., Constable, I. J., Mellough, C. B., Carvalho, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelium+and+age-related+macular+degeneration:+A+review+of+major+disease+mechanisms.">Retinal pigment epithelium and age-related macular degeneration: A review of major disease mechanisms.</a> Clinical &amp; Experimental Ophthalmology. 48 (8), 1043-1056 (2020).</li><li>Xu, H. Z., Song, Z., Fu, S., Zhu, M., Le, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE+barrier+breakdown+in+diabetic+retinopathy:+seeing+is+believing.">RPE barrier breakdown in diabetic retinopathy: seeing is believing.</a> Journal of Ocular Biology, Diseases, and Informatics. 4 (1-2), 83-92 (2011).</li><li>Hellinen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+artificially+re-pigmented+ARPE-19+retinal+pigment+epithelial+cell+model.">Characterization of artificially re-pigmented ARPE-19 retinal pigment epithelial cell model.</a> Scientific Reports. 9 (1), 13761 (2019).</li><li>Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+of+the+human+RPE+cell+line,+ARPE-19,+induced+by+nicotinamide.">Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.</a> Experimental Eye Research. 179, 18-24 (2019).</li><li>Samuel, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriately+differentiated+ARPE-19+cells+regain+phenotype+and+gene+expression+profiles+similar+to+those+of+native+RPE+cells.">Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.</a> Molecular Vision. 23, 60-89 (2017).</li><li>Middleton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+ophthalmology.">Porcine ophthalmology.</a> Veterinary Clinics of North America: Food Animal Practice. 26 (3), 557-572 (2010).</li><li>Farjood, F., Ahmadpour, A., Ostvar, S., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+mechanical+stress+in+primary+porcine+RPE+cells+induces+angiogenic+factor+expression+and+in+vitro+angiogenesis.">Acute mechanical stress in primary porcine RPE cells induces angiogenic factor expression and in vitro angiogenesis.</a> Journal of Biological Engineering. 14, 13 (2020).</li><li>Fietz, A., Hurst, J., Joachim, S. C., Schnichels, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+primary+porcine+retinal+pigment+epithelium+monolayer+to+complement+retinal+ex+vivo+cultures.">Establishment of a primary porcine retinal pigment epithelium monolayer to complement retinal ex vivo cultures.</a> STAR Protocols. 4 (3), 102443 (2023).</li><li>Hood, E. M. S., Curcio, C. A., Lipinski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+cryosectioning+of+primary+porcine+retinal+pigment+epithelium+on+transwell+cell+culture+inserts.">Isolation, culture, and cryosectioning of primary porcine retinal pigment epithelium on transwell cell culture inserts.</a> STAR Protocols. 3 (4), 101758 (2022).</li><li>Toops, K. A., Tan, L. X., Lakkaraju, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+three-step+protocol+for+live+imaging+of+intracellular+traffic+in+polarized+primary+porcine+RPE+monolayers.">A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers.</a> Experimental Eye Research. 124, 74-85 (2014).</li><li>Rickabaugh, E., Weatherston, D., Harris, T. I., Jones, J. A., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+biomimetic+in+vitro+model+of+bruch's+membrane+using+hagfish+slime+intermediate+filament+proteins.">Engineering a biomimetic in vitro model of bruch's membrane using hagfish slime intermediate filament proteins.</a> ACS Biomaterials Science &amp; Engineering. 9 (8), 5051-5061 (2023).</li><li>Harris, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+recombinant+spider+silk+proteins+to+develop+a+synthetic+bruch's+membrane+for+modeling+the+retinal+pigment+epithelium.">Utilizing recombinant spider silk proteins to develop a synthetic bruch's membrane for modeling the retinal pigment epithelium.</a> ACS Biomaterials Science &amp; Engineering. 5 (8), 4023-4036 (2019).</li><li>Zou, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+tight+junction+between+RPE+cells+with+tissue+factor+targeting+peptide.">Protection of tight junction between RPE cells with tissue factor targeting peptide.</a> International Journal of Ophthalmology. 11 (10), 1594-1599 (2018).</li></ol>

This protocol describes how to isolate RPE cells from porcine eyes. Pigmentation and cobblestone morphology are seen within 7 days of isolation (Figure 1B). Furthermore, TEER data indicate tight junction formation22 and a healthy monolayer (Figure 5). These results show that RPE cells isolated with this method are similar to human RPE and can be beneficial in retinal cell culture models.
The eyes used in this m...

The retinal pigment epithelium (RPE) is a monolayer located in the outer retina&#160;between Bruch&#39;s membrane and the photoreceptors1. RPE cells form tight junctions with proteins such as zonula occludens-1 (ZO-1) and possess a distinctive phenotype characterized by pigmentation and hexagonal morphology2,3. These cells contribute to the blood-retinal barrier, thereby supporting photoreceptor health and maintaining retinal homeostasis4,5. Additionally, RPE cells play a critical role in vision by absorbing light and recycling essential components for the photoreceptors6. For instance, RPE65, a protein highly expressed in RPE cells, converts all-trans retinyl esters to 11-cis retinol7,8. Given the multitude of functions performed by RPE cells, their dysfunction is implicated in various diseases, including age-related macular degeneration and diabetic retinopathy9,10. To enhance the understanding of retinal pathologies and develop new treatments, in vitro models of the retina are frequently employed.
To generate representative models of healthy or diseased retinas, it is imperative to use a mimetic RPE cell type. The commercially available ARPE-19 cell line lacks native phenotypes, such as pigmentation, while iPSCs can take months to differentiate11,12,13. Although human donor eyes may be ideal, they are often not readily available to many research labs.
Here, we have devised a method to utilize porcine eyes, which share many similarities with human eyes14, for obtaining primary RPE cells. These primary porcine RPE cells have been utilized in multiple retinal models15,16. Not only are these cells cost-effective, but they also require less time to acquire than iPSCs or donor eyes. Additionally, they exhibit native characteristics, such as pigmentation and microvilli. While similar protocols for porcine RPE extraction exist17,18,19, this straightforward and detailed technique further validates enzymatic dissociation and employs materials commonly found in most cell culture laboratories.

<table><tbody><tr><td>6 Micro-well glass bottom plate with 14 mm micro-well #1 cover glass</td><td>Cellvis</td><td>P06-14-1-N</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>Calcium Chloride, Dried, Powder, 97%</td><td>Alfa Aesar</td><td>L13191.30</td><td></td></tr><tr><td>Cell Strainer</td><td>Fisher Scientific&amp;nbsp;</td><td>22-363-548</td><td>one per eye</td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Centrifuge Tubes, 15 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>05-539-12</td><td></td></tr><tr><td>Cooler, 8 L</td><td>Igloo</td><td>32529</td><td></td></tr><tr><td>Corning&amp;nbsp;Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts</td><td>Fisher Scientific&amp;nbsp;</td><td>07-200-154</td><td>Culture inserts</td></tr><tr><td>Cut Resistant Glove</td><td>Dowellife</td><td>712971375857</td><td></td></tr><tr><td>Cytiva&amp;nbsp;HyClone Dulbecco&#039;s Phosphate Buffered Saline, Solution</td><td>Fisher Scientific&amp;nbsp;</td><td>SH3026401</td><td>for ICC dilutions only&amp;nbsp;</td></tr><tr><td>Deionized Water</td><td></td><td></td><td></td></tr><tr><td>DMEM, 1x with 4.5 g/L glucose, L-glutamine &amp;amp; sodium pyruvate</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>DNase I from Bovine Pancreas</td><td>Sigma Aldrich</td><td>DN25</td><td></td></tr><tr><td>DPBS/Modified - calcium - magnesium</td><td>Cytiva</td><td>SH3002B.02</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>ELISA kit, Q-Plex Human Angiogenesis (9-Plex)&amp;nbsp;</td><td>Quansys Biosciences, Logan, UT</td><td></td><td></td></tr><tr><td>ENDOHM 6 TEER device</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Avantor</td><td>232B20</td><td></td></tr><tr><td>Fisher BioReagents&amp;nbsp;Bovine Serum Albumin (BSA) DNase- and Protease-free Powder</td><td>Fisher Scientific&amp;nbsp;</td><td>BP9706100</td><td></td></tr><tr><td>Flashlight</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde, ACS Grade, 36.5% (w/w) to 38.0% (w/w), LabChem</td><td>Fisher Scientific&amp;nbsp;</td><td>LC146501</td><td></td></tr><tr><td>Gauze Sponges</td><td>Fisher Scientific&amp;nbsp;</td><td>22-415-504</td><td>One per eye</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, Invitrogen</td><td>Thermo Scientific</td><td>A32728</td><td>RPE65 secondary antibody</td></tr><tr><td>Ice&amp;nbsp;</td><td></td><td></td><td>Crushed prefered</td></tr><tr><td>Inverted Phase Contrast Microscope</td><td></td><td></td><td></td></tr><tr><td>Invitrogen&amp;nbsp;NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td><td>Fisher Scientific&amp;nbsp;</td><td>R37605</td><td></td></tr><tr><td>Iris Fine Tip Scissors, Standard Grade, Curved, 4.5&quot;</td><td>Cole-Palmer</td><td>EW-10818-05</td><td></td></tr><tr><td>Iris Scissors, 11 cm, Straight, Tungsten Carbide</td><td>Fisher Scientific&amp;nbsp;</td><td>50-822-379</td><td></td></tr><tr><td>LSM-710 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Petri Dish, 100 mm x 20 mm&amp;nbsp;</td><td>Corning</td><td>430167</td><td>one per 2-3 eyes and one for dissection surface/waste&amp;nbsp;</td></tr><tr><td>Povidone-Iondine Solution, 10%</td><td>Equate</td><td>49035-050-34</td><td></td></tr><tr><td>RPE65 Monoclonal Antibody (401.8B11.3D9), Invitrogen</td><td>Thermo Scientific</td><td>MA116578</td><td>RPE65 primary antibody</td></tr><tr><td>Scalpel Blades Size 10</td><td>Fisher Scientific&amp;nbsp;</td><td>22-079-683</td><td></td></tr><tr><td>Scalpel Handles Style 3</td><td>Fisher Scientific&amp;nbsp;</td><td>50-118-4164</td><td></td></tr><tr><td>Surgical Drape, 18 x 26&quot;</td><td>Fisher Scientific&amp;nbsp;</td><td>50-209-1792</td><td></td></tr><tr><td>Tissue Culture Incubator</td><td></td><td></td><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, 95% Humidity</td></tr><tr><td>Tissue Culture Plates, 6 Wells</td><td>VWR</td><td>10062-892</td><td>One for eye wash and one for seeding&amp;nbsp;</td></tr><tr><td>Tri-Cornered Polypropylene Beaker, 1000 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>14-955-111F</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Trypsin 0.25%, 2.21 mM EDTA in HBSS; w/o Ca, Mg, Sodium Bicarbonate</td><td>Corning</td><td>25053Cl</td><td></td></tr><tr><td>Tweezers Style 20A</td><td>Fisher Scientific&amp;nbsp;</td><td>17-467-231</td><td></td></tr><tr><td>Tweezers Style 2A</td><td>Fisher Scientific&amp;nbsp;</td><td>50-238-47</td><td>for removing neural retina</td></tr><tr><td>Tweezers Style 5-SA-PI</td><td>Fisher Scientific&amp;nbsp;</td><td>17-467-168</td><td></td></tr><tr><td>Vacuum Aspiration System</td><td></td><td></td><td></td></tr><tr><td>Water Bath, 37 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>ZO-1 Monoclonal Antibody (ZO1-1A12), FITC, Invitrogen</td><td>Fisher Scientific&amp;nbsp;</td><td>33-911-1</td><td>ZO-1 conjugated primary antibody</td></tr></tbody></table>

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.

Using this procedure, primary RPE cells were successfully isolated from porcine eyes. Figure 1A shows RPE cells 3 days post isolation with characteristic pigmentation. After 1 week of growth, cells were fully confluent and formed a healthy monolayer (Figure 1B). Cells were then transferred to cell culture inserts where they maintained their pigmentation and morphology (Figure 1C), further supporting the efficacy of the isolation pro...

Watch this Scientific Journal Video about Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models at JoVE.com

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.

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 ...

isolation-primary-porcine-retinal-pigment-epithelial-cells-for-vitro

Research

JoVE Journal

Biology

907 Views.  Utah State University. 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.

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neuroscience

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 ...

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 ...

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.

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 ...

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 ...

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. ...

Leporine

Isolation of IPE and RPE cells: A Technique to Obtain Pigmented Epithelial Cells from Isolated Porcine 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 describe a technique to isolate iris and retinal pigment epithelial cells from porcine eyes to obtain their primary cultures for in vivo and ex vivo studies. These pigment cells could be genetically modified to study regenerative approaches to treat ocular disorders.

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