This work was supported by the Ocular Genomics Institute at Massachusetts Eye and Ear.

The guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research were followed.
NOTE: This method has been proven successful with mice of different genetic backgrounds, including C57BL/6J, B10.D2-Hco H2d H2-T18c/oSnJ, and albino mice, at various ages. Preferably use 8 to 12-week-old mice to obtain RPE cells. RPE cells from older mice proliferate less in culture and younger mice have fewer and smaller cells, which requires pooling eyes from different animals to have viable cultures.
1. Preparation of reagents and the membrane inserts
<ol>
	<li>Prepare the following reagents.
	<ol>
		<li>Prepare HBSS-H&#8722; (HBSS without calcium, without magnesium buffer + 10 mM HEPES): Add 1 mL of 1 M HEPES to 99 mL of HBSS- (without Ca/Mg) buffer. Store at 4 &#176;C up to 1 month.</li>
		<li>Prepare HBSS-H+ (HBSS with calcium, with magnesium buffer + 10 mM HEPES): Add 1 mL of 1 M HEPES to 99 mL of HBSS+ (with Ca/Mg) buffer. Store at 4 &#176;C up to 1 month.</li>
		<li>Prepare hyaluronidase solution (1 mg/mL): Add 10 mg of hyaluronidase to 10 mL of HBSS-H&#8722; and filter it with a 0.4 &#181;m sterile syringe filter using a 10 mL syringe. Prepare fresh before use and leave it at room temperature (RT; 20-25 &#176;C).</li>
		<li>Prepare FBS solution: Mix 20% FBS with HBSS-H+. Add 2 mL of FBS to 8 mL of HBSS-H+. Prepare fresh before use.</li>
		<li>Prepare RPE medium: N1 Medium Supplement 1/100 (v/v), glutamine 1/100 (v/v), penicillin-streptomycin 1/100 (v/v) and nonessential amino acid solution 1/100 (v/v), hydrocortisone (20 &#181;g/L), taurine (250 mg/L) and triiodo-thyronin (0.013 &#181;g/L) in alpha MEM + 5% FBS or without FBS1,23,24. If desired, FBS may be heat-inactivated; no differences have been observed. Prepare fresh for RPE isolation. For cell culture maintenance, store at 4 &#176;C up to 1 month.</li>
		<li>Prepare trypsin-EDTA: Prepare small single-use aliquots (~8 mL) of fresh trypsin-EDTA (0.25%) and freeze them at -20 &#7506;C. Thaw them at RT before each use. Avoid freeze-thaw cycles.</li>
	</ol>
	</li>
	<li>Prepare the membrane inserts (e.g., Transwell inserts).
	<ol>
		<li>Equilibrate the 6.5-mm membrane inserts with RPE medium for at least 30 min at 37 &#176;C, in a 5% CO2-aerated incubator. Use one membrane insert to seed RPE cells from two mouse eyes.</li>
		<li>After incubation, replace the medium of the lower compartment with 700 &#181;L of PBS.</li>
		<li>Remove the medium from the top compartment and coat the membrane insert with 100 &#181;L of 10 &#181;g/mL mouse laminin (in PBS) for at least 2 h at RT (shorter incubations may lead to poor attachment of the cells to the insert).</li>
		<li>Coat an extra membrane insert to use as a blank for TER measurements.</li>
	</ol>
	</li>
</ol>
2. Dissection and enucleation of the mouse eyes
<ol>
	<li>Euthanize the mice by CO2 asphyxiation by placing them in a CO2 chamber and slowly releasing CO2 at a fill rate of 30-70% of the chamber volume per minute.</li>
	<li>Place the angled, serrated tips of micro-forceps on each side of the mouse eye and gently press to proptose the eyeball (enucleation).</li>
	<li>Close the forceps placed around the eyeball. Then, pull gently while moving forward and backward to detach the whole eye with the optic nerve from the ocular muscles, ensuring that no connective tissue remains attached to the sclera.</li>
	<li>Rinse the enucleated eyeball in 70% ethanol before placing them into one well of a six-well plate with 3 mL of HBSS-H&#8722; on ice.</li>
	<li>Repeat steps 2.2 to 2.4 to remove the second eye of the mouse. Proceed with the next steps within 30 min. 
	NOTE: It is recommended enucleating/dissecting only two eyes at a time until some experience is acquired.</li>
</ol>
3. Collection of the RPE
NOTE: Perform the following steps under sterile conditions in a laminar flow hood. To avoid extended incubations on ice, which can result in RPE cell death, do not collect more than two eyes at a time.
<ol>
	<li>Use a dissecting stereomicroscope, Dumont #5 forceps, and angled scissors to carefully clean away all the connective tissue, blood and muscles remaining attached to the eyeball without making any cuts in the sclera. Change the 3 mL of HBSS-H&#8722; buffer regularly, as needed, to keep the eye fresh and clean, and avoid the contamination of the RPE cultures.</li>
	<li>Use the optic nerve as a handle to hold the eyeball and make a hole in the center of the cornea with a sharp carbon-steel #11 blade.</li>
	<li>Use Dumont #5 scissors to make three incisions in the cornea through the aforementioned hole, ensuring that there is sufficient space to remove the lens.</li>
	<li>Hold the optic nerve and apply slight pressure to the ora serrata with the base of the angled scissors until the lens comes completely out. Leave the iris epithelium in place to prevent the detachment of the neural retina and RPE during incubation. Place the eye in HBSS-H-buffer.</li>
	<li>Repeat Steps 3.1-3.4 to dissect the second eye.</li>
	<li>Incubate the eyes without lenses in hyaluronidase solution in a 12-well plate at 37 &#176;C for 45 min in a 5% CO2-aerated incubator (1.5 mL/well) to detach the neural retina from the RPE.</li>
	<li>Place each eye in a new well and incubate it on ice for 30 min with 1.5 mL of cold HBSS-H+ buffer per well to stop the hyaluronidase activity. Do not extend the incubation for longer than 45 min.</li>
	<li>Wash, and place each eye into a 35-mm culture dish with fresh HBSS-H+ buffer and cut the cornea through the original incisions until reaching the the ora serrata&#160;by using 8-cm Vannas scissors. Then, cut below the ora serrata to remove the iris epithelium and cornea.</li>
	<li>Hold the eyecup edge/ora serrata with curved tweezers and, using angled microforceps, pull away the neural retina making sure that the RPE layer is not cut. Then, cut the internal attachment to the optic nerve. If some RPE cells remain attached to the neural retina, extend the incubation time but do not exceed 45 min total.</li>
	<li>Cut the optic nerve and transfer each eyecup to a different 12-well plate containing 1.5 mL of fresh trypsin-EDTA per well. Ensure that the eyecups remain opened and completely submerged in the trypsin. Incubate the eyecups at 37 &#176;C for 45 min in a 5% CO2 incubator.</li>
	<li>Collect each eyecup together with any RPE sheets detached during the trypsin incubation and transfer them into a 12-well plate containing 1.5 mL of FBS solution per well. If RPE sheets remain in the trypsin solution, use a micropipette to transfer them to the well with FBS solution.</li>
</ol>
4. Isolation of primary mouse RPE cells
<ol>
	<li>Hold each eyecup by the optic nerve and shake it face down into the 12-well plate containing 1.5 mL of 20% FBS in HBSS-H+ until the complete detachment of the RPE sheets is achieved.</li>
	<li>Collect any RPE sheets and RPE clusters with a micropipette and place them in a 15-mL tube. Avoid any white pieces of sclera or choroid, which could contaminate the cultures. Pool two eyes from the same mouse in one tube.</li>
	<li>Centrifuge the mixture at 340 x g for 2 min at RT and discard the supernatant.</li>
	<li>Gently resuspend the RPE pellet in 1 mL of trypsin-EDTA (0.25%) and incubate the mixture for 1 min in a water bath at 37 &#176;C to disaggregate the RPE sheets into single cells. After incubation, gently pipet up and down 10x with a micropipette, avoiding bubble formation while pipetting.</li>
	<li>Add 9 mL of freshly prepared RPE medium to dilute and inactivate the trypsin and centrifuge the mixture at 340 x g for 2 min at RT.</li>
	<li>Aspirate the supernatant and carefully resuspend the cell pellet in 150 &#181;L of RPE medium with 5% FBS by using a micropipette, ensuring that cells are homogeneously resuspended and avoiding bubble formation while pipetting.</li>
	<li>Take laminin-coated membrane insert from step 1.2, remove PBS from the bottom chamber and add 700 &#181;L of RPE medium.</li>
	<li>Remove the laminin from the upper chamber of the membrane insert and distribute the RPE cell suspension dropwise and uniformly to the center of the chamber, avoiding bubble formation while pipetting.</li>
	<li>Place the membrane insert in a 5% CO2 incubator at 37 &#176;C and leave undisturbed for at least 24 h.</li>
	<li>After 24 h, check the membrane insert under the microscope to make sure that most RPE cells are attached to the insert and the confluence is at least 50% (Figure 1A). It is critical not to change media during the first 72 h.</li>
</ol>
5. Culture of polarized RPE monolayers
<ol>
	<li>Maintain the isolated RPE cells in culture for at least 72 h before refreshing the RPE medium to allow cell attachment. A cell confluence of 50% or more at seeding is fundamental for the formation of a suitable polarized RPE monolayer.</li>
	<li>Change the culture medium twice a week using fresh and pre-warmed RPE medium (step 1.1.5) after the cells are attached. Serum can be removed from the culture medium after the first 72 h. 
	&#8203;NOTE: After one week in culture, cells should be confluent, hexagonal, bi-nucleated, pigmented and polarized (Figure 1B), with expected cell numbers being around 50,000 cells per 6.5 mm Transwell insert. After two weeks in culture, RPE monolayers comprising of healthy cells are observed. RPE cultures display apical microvilli, basal infoldings, and tight junctions (Figure 1C).</li>
</ol>
6. TER measurement
NOTE: Perform TER measurements of the RPE cells after a minimum of 4 days in culture to ensure a good integrity and polarization of the RPE monolayer.
<ol>
	<li>Clean the electrodes of the voltohmmeter with 70% ethanol and dry them carefully.</li>
	<li>Take the transwells from the incubator and perform TER measurement within 3 min to avoid alterations due to temperature fluctuations. To measure TER, immerse the short electrode of the voltohmmeter in the upper chamber and the long electrode in the bottom chamber of the membrane insert. Avoid contact with the RPE monolayer to prevent cell detachment.</li>
	<li>To calculate TER, deduct the value of the blank (membrane insert coated with laminin without cells) from the sample. Then multiply the obtained value (in ohms) by the surface area of the membrane insert (0.33 cm2 in the case of 6.5-mm membrane insert). The product should be at least 200 &#8486; x cm2 after 72 h in culture. TER values should measure above 400 &#8486; x cm2 after two weeks. Discard RPE cultures with low TER values.</li>
</ol>

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The authors declare that they have no competing financial interests.

While several methods for mouse RPE cell isolation and culture had been developed before1,13,20,22,26,27, Fernandez-Godino&#39;s method first used membrane inserts allowing the efficient growth of the RPE cells in culture for weeks1,9. Another major change in their pr...

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse retinal pigment epithelium (RPE) cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The&#160;RPE&#160;is a monolayer located in the eye between the neural retina and the Bruch&#39;s membrane. This single layer consists of highly polarized and pigmented epithelial cells joined by tight junctions, exhibiting a hexagonal shape that resembles a honeycomb2. Despite this apparent histological simplicity, the RPE performs a wide variety of functions critical to the retina and the normal visual cycle2,3,4. The main functions of the RPE monolayer include light absorption, nourishment and renewal of photoreceptors, removal of metabolic end products, control of the ion homeostasis in the subretinal space and maintenance of the blood-retinal barrier2,3. The RPE also has an important role in local modulation of the immune system in the eye5,6,7,8,9,10,11. Degeneration and/or dysfunction of the RPE are common features shared by many ocular disorders such as retinitis pigmentosa, Leber congenital amaurosis, albinism, diabetic retinopathy, and macular degeneration12,13,14,15. Unfortunately, the availability of human tissues is limited. Given their highly conserved genetic homology with humans, mouse models represent a suitable and useful tool for studying ocular disorders16,17,18,19. Furthermore, the use of cultured primary RPE cells provides advantages such as genetic manipulation and drug testing that can accelerate the development of new therapies for these vision-threatening disorders9,11.
Existing methods available for mouse RPE isolation and culture lack reproducibly and do not recapitulate the RPE features in vivo with enough reliability. Cells tend to lose pigmentation, hexagonal shape and transepithelial electrical resistance (TER) within a few days in culture13, 20. Since establishing these primary RPE cell cultures from mice is a challenging process, this optimized protocol has been created based on other protocols to isolate RPE cells from rat and human eyes21,22,23 to dissect the mouse eyes, collect the RPE and culture the mouse RPE cells in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 ml BD Luer-Lok tip syringe, disposable</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>VWR International</td>
			<td>21008-103</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>VWR International</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M4526-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled micro forceps</td>
			<td>WPI</td>
			<td>501727</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench-top centrifuge</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo</td>
			<td>HERA VIOS 160I CO2 SST TC 120V</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phospate Buffered Saline no Calcium, no Magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 45&#176; Medical Biology tweezers, 0.05 x 0.01 mm tip, 11 cm length</td>
			<td>WPI</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Clear Polystyrene Cell Culture Dish, 35mm</td>
			<td>BD Biosciences</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td>Heat inactivated.</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red</td>
			<td>Life Technologies</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red B6</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Gibco</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H-3506 1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H-0396</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo</td>
			<td>CLASS II A2 4 115V PACKAGECLA</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin 1mg/ml</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1 MG</td>
			<td>Dilute in PBS at 37C to 1mg/ml</td>
		</tr>
		<tr>
			<td>McPherson-Vannas Micro Scissors 8 cm long</td>
			<td>WPI</td>
			<td>503216</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids 100X</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 Supplement 100X</td>
			<td>Sigma-Aldrich</td>
			<td>N6530-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Bard-Parker Carbon steel surgical blade size 11</td>
			<td>Fisher-Scientific</td>
			<td>08-914B</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated 12-well plates</td>
			<td>Fisher-Scientific</td>
			<td>08-772-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated 6-well plates</td>
			<td>Fisher-Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell supports 6.5 mm</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3470-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>Sigma-Aldrich</td>
			<td>T-5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer, Dumont #5 Medical Biology 11 cm, curved, stainless steel 0.02 x 0.06 mm Mod tips</td>
			<td>WPI</td>
			<td>500232</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors 8cm long, stainless steel</td>
			<td>WPI</td>
			<td>501790</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Puradisc 25mm Syringe Filters 0.45&#956;m pore size</td>
			<td>Fisher-Scientific</td>
			<td>6780-2504</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient dissection and culture of primary mouse retinal pigment epithelial cells

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

This protocol has been used to isolate and culture RPE cells from genetically modified mice1. No differences have been observed between mouse strains or gender. The results have helped to understand some important aspects of the mechanism underlying ocular diseases such as age-related macular degeneration, which is the most common cause of vision loss among the elderly9. RPE cells isolated following this protocol were completely attached to the membrane insert 24 hours afte...

Watch this Scientific Journal Video about Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells at JoVE.com

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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

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

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6.1K Views.  Harvard Medical School. This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Video: Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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 ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

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 ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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 ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...