Name: primary cell cultures from the mouse retinal pigment epithelium
Uploaded: 2018-03-16T13:30:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

This study was partly funded by The BrightFocus Foundation (to DS).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at&#160;University of Pittsburgh
1. Prepare solutions
<ol>
	<li>Prepare growth medium by supplementing Dulbecco Modified Eagle Medium (DMEM), high glucose with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2.5 mM L-glutamine, and 1x MEM nonessential amino acids. Pre-warm the media in 37 &#176;C incubator before use.</li>
	<li>Prepare 2% (wt/vol) dispase II working solution:
	<ol>
		<li>Prepare a stock solution of 100 mg/mL dispase II by dissolving 30 mg of dispase II in 300 &#181;L HEPES-buffered saline (50 mM HEPES/KOH pH 7.4, 150 mM NaCl). This volume is for 3 - 4 mice, it can be scaled up or down based on the number of mice for the experiment). This stock can be stored for at least 1 week at 4 &#176;C.</li>
		<li>Prepare the working solution of 2% dispase by adding 300 &#181;L of the 100 mg/mL dispase II stock to 1.2 mL of sterile DMEM, high glucose and filtering the solution through a 0.22 &#181;m filter. This step should be performed in the laminar flow cabinet under sterile conditions. Pre-warm the prepared dispase II in 37 &#176;C incubator before use.</li>
	</ol>
	</li>
</ol>
2. Obtaining the mouse eyes
<ol>
	<li>Euthanize the mouse using any approved method and place it on an absorbent pad.</li>
	<li>Wearing gloves, put the thumb and index finger around the eye and gently push the skin to proptose the eyeball.</li>
	<li>Insert the tip of angled scissors between the skin and the eyeball and carefully cut out the eye. To avoid contamination, briefly dip the eye in 70% ethanol.</li>
	<li>Immediately place the eye in the Phosphate-buffered Saline (PBS) in a Petri dish.</li>
	<li>Repeat steps 2.2 - 2.4 to remove the second eye.</li>
</ol>
3. Eye dissection
<ol>
	<li>Carefully remove connective tissue from the eye under the dissecting microscope. This can be done either in PBS or on the dry Petri dish.
	<ol>
		<li>Use suturing forceps to lift the connective tissues from the eyeball and cut them out using Vannas scissors. Do not cut the sclera because it is practically impossible to obtain the intact posterior eyecups if the sclera is cut.</li>
		<li>After cleaning the connective tissue, keep the resulting eyeballs in PBS for the next washing step. 
		NOTE: All the following steps should be performed in the laminar flow cabinet under sterile conditions.</li>
	</ol>
	</li>
	<li>Wash the eyes twice in pre-warmed 37 &#176;C DMEM (high glucose). Carefully transfer the eyeballs into the pre-warmed 37 &#176;C DMEM (high glucose) using forceps. Aspirate the medium and transfer eyeballs to fresh medium in a new dish.</li>
	<li>Aspirate DMEM (high glucose) medium and incubate the eyes in the pre-warmed 2% (wt/vol) dispase II working solution for 45 min in the 37 &#176;C incubator. 
	NOTE: In all the following steps, place and manipulate eyes or eyecups in the growth medium, which will protect the tissue and provide a good medium for the dissection.</li>
	<li>Remove the dispase II solution and wash the eyes twice in growth medium by aspirating the old medium and adding the new pre-warmed 37 &#176;C growth medium (Figure 1a). 
	NOTE: After dissociation by dispase II, the eyeballs become soft.</li>
	<li>Hold the eye with forceps and make an incision around the ora serrata of each eye using Vannas scissors under the dissecting microscope placed in the laminar flow cabinet.
	<ol>
		<li>Keeping the eyeball in the solution, carefully remove the anterior cornea by extending the cut around the ora serrata circumference and pulling the anterior cornea away after the cut has been complete.</li>
		<li>Remove the lens capsule and associated iris pigmented epithelium by gently pulling them out of the eyecup with teethed forceps (Figure 1b, 1c).</li>
	</ol>
	</li>
	<li>Incubate the resulting posterior eyecups in the growth medium for 20 min at 37 &#176;C to facilitate separation of the neural retina from the RPE.</li>
	<li>Cut the posterior eyecups into 4 petals under the dissecting microscope placed in the laminar flow cabinet. The cuts should be long enough to flatten the eyecup, but short enough to keep the petals connected (Figure 1d).
	<ol>
		<li>Flatten the eyecup and remove the neural retina by very gently pulling it with forceps while holding the remaining RPE-choroid-sclera complex with another forceps (Figure 1e). Start pulling from the edges rather than from the center.</li>
	</ol>
	</li>
	<li>Transfer all the quartered tissues into a new sterile culture dish filled with pre-warmed 37 &#176;C growth medium.</li>
</ol>
4. Isolation of primary RPE cells
<ol>
	<li>Holding one petal of the quartered RPE-choroid-sclera complex with super fine forceps, gently peel off the intact sheets of RPE from the underlying basement membrane (Bruch&#39;s membrane) by using a microsurgical crescent knife under the dissecting microscope placed in the laminar flow cabinet (Figure 1f).
	<ol>
		<li>Transfer the RPE sheets into the new sterile culture dish containing growth medium by using a P200 micropipette. When transferring RPE sheets, wet the pipette tips with growth medium by pipetting several times to prevent tissue from sticking inside the tip. RPE will be clearly distinguishable from the choroid: RPE looks more brownish, while choroid is darker and looks sticky and fluffy.</li>
		<li>Alternatively, use enzymatic digestion and gentle dissociation without forceps to detach the RPE from the choroid14.</li>
	</ol>
	</li>
	<li>Wash the RPE sheets 2 - 3 times using pre-warmed 37 &#176;C growth medium in the sterile culture dish. After each washing step, carefully collect the RPE sheets while avoiding other tissues such as retina debris or choroid. At the end of the last wash collect the RPE sheets with a P200 micropipette and place them in a new 1.5 mL microcentrifuge tube. 
	NOTE: There is no need to centrifuge the RPE sheets: they will sediment by gravity within 1 min.</li>
	<li>Remove the extra growth medium using a P200 micropipette. Resuspend the cells in fresh pre-warmed growth medium and gently triturate them using a P200 micropipette. Avoid formation of bubbles. A single-cell suspension is ideal, but also avoid too much trituration, otherwise RPE cells are not able to survive. We recommend very gently trituration for 40 - 50 times.</li>
</ol>
5. Culturing RPE cells
<ol>
	<li>Plate the cells on a culture plate.
	<ol>
		<li>If downstream applications of the RPE cells require retaining of their polarity, plate the RPE cells from 2 mice&#160;in one 12 mm polyester membrane insert pre-loaded in 12-well culture plates. Plating the cells at high density is desired because in that case RPE cells are able to keep the original properties, such as hexagonal shape and pigmentation.</li>
	</ol>
	</li>
	<li>Do not move the plate or change the growth medium for the first 72 h of culturing. After 3 days, replace the old medium with the fresh pre-warmed growth medium. After that change the culture medium every other day. Once RPE cells get confluency, reduce the FBS in growth medium to 2%. 
	NOTE: The cells can be split using 0.25% trypsin. However, after splitting the cells may lose their hexagonal shape and pigmentation in the subsequent passages. We do not recommend splitting the cells, but if it is required by the downstream applications of the culture, keep in mind that the primary cells cannot be passaged indefinitely: they are known to de-differentiate after 5 - 7&#160;passages.</li>
</ol>

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DS has received research funding from Bayer HealthCare, Germany and F. Hoffmann-La Roche, Switzerland. The remaining authors declare no competing financial interests.&#160;This work is supported by Research to Prevent Blindness (unrestricted grants to the Wilmer Eye Institute and University of Pittsburgh).&#160; This work is also supported by start-up funds to DS from Ophthalmology, University of Pittsburgh.

The presented detailed protocol enables reliable establishment of mouse primary RPE cultures that reach confluency after 1 week and present the main RPE characteristics such as hexagonal shape and pigmentation after 2 weeks. The obtained RPE cells can be used for a number of downstream applications such as vitamin A metabolism9, phagocytosis of the photoreceptor outer segments10 and ion transport11, epithelial cell polarity2, lysosomal homeostasis, and autophagy12,13. Depending on the downstream application, the morphology of the derived cultures can be validated by transmission and scanning electron microscopy as described in detail elsewhere14. Additional assays to demonstrate maturation of the cultures might include transepithelial electrical resistance (TEER) assay to demonstrate polarity of the RPE cells, permeability assays4,22, functional tests (phagocytosis23) and dome formation.
In our experience, the more cells that are plated in a well, the better the confluency and the phenotype. In this protocol, we seeded the RPE cells from 2 mice on one 12 mm inserts. However, it is also feasible to put RPE cells from 2 mice into one 6.5 mm insert to get better confluency and TEER. &#8203;In many studies, the plates for RPE cell cultures are coated with Matrigel or laminin. We have not seen a significant difference in RPE cell attachment to these vessels with or without coating. In addition, the percentage of FBS in the culture medium might be modified to achieve better results. 10% FBS is usually good for reaching confluency, but dropping to less FBS seems to be beneficial for RPE differentiation/maturation14,17.
The main limitation of this method is the need for precise training of the person performing the eye dissections to ensure maximum efficiency of RPE cell recovery without cross-contamination with cells derived from other ocular tissues. To avoid cross-contamination, the researcher should master the critical step of this procedure: careful peeling off the retina followed by precise separation of the RPE from the choroid. The purity of the obtained cultures can be validated using RPE-specific immunostaining. A number of RPE markers are available for characterization of the RPE cultures, including genes involved in the visual cycle, barrier and transport function, metabolism, phagocytosis, and melanogenesis15.
Another limitation is the number of eyes that can be processed simultaneously to obtain the cultures. In general, we do not recommend working with more than 2 mice at a time. To ensure viability of the RPE cells, the eyes should be dissected as quickly as possible. After the user become more proficient, it is possible to handle more mice by alternating incubation times.
Overall, a number of methods have been published on the establishing the primary mouse RPE cultures3,14,15, however, for the first time, we present a video version of the complete protocol. This protocol can be used as guidance on the eye dissection techniques. However, the optimal balance between the high speed and precision required for the dissection can be achieved only with practice.

The retinal pigment epithelium (RPE) is a single layer of polarized epithelial cells that is located between the neural retina and the choroid of the eye. The functionality of the RPE cells and the integrity of the RPE monolayer are critical for vision because the RPE plays a major role in multiple processes such as maintaining the outer blood-retinal barrier, transport of water and ions between the retina and the choroid, light absorption, protection from oxidative stress, control of retinoid metabolism, and phagocytosis of the outer segments of the photoreceptors1,2. The location of the RPE at the back of the eye, as well as its barrier function preventing drugs administered systemically from passing from the blood to the vitreous humor, make it difficult to study the complexity of the RPE function in vivo. Thus, there is a great need for the establishment of RPE cell cultures to study the RPE cells in a flexible, controlled environment3,4.
A number of established RPE cell lines exist, providing an easy and convenient way of obtaining and storing the cells; however, passaged cells have some disadvantages compared to primary cells2,3,4. First, they are often characterized by changes in cell morphology. For example, none of the existing cell lines were found to be suitable for a reliable study of the RPE barrier properties due to the loss of cell polarity phenotype and partial disappearance of tight junctions4. In addition to the loss of polarity and proper cell-to-cell connections, the RPE cell lines quickly lose their pigmentation due to the absence of the key melanogenesis enzymes in the adult RPE5. The pigmentation can be restored, but the comprehensive analysis of the mechanism of re-pigmentation which would include a combination of transmission electron microscopy, gene expression analysis and chemical assays to confirm the presence of melanin has never been performed6. One more limitation is that the RPE cell lines have extended cell life (sometimes - immortality) and under certain conditions can transform into self-renewing multipotent stem cells that detach from the substrate and form floating colonies7,8. This limitation makes it impossible to use the cell lines for transplantation experiments3.
Considering the disadvantages of the established RPE cell lines, primary RPE cell cultures obtained from fresh tissues might serve as a more biologically relevant model to study the RPE. Primary RPE cells have been used not only to study RPE-specific functions such as vitamin A metabolism9, phagocytosis of the photoreceptor outer segments10 and ion transport11, but also to study basic cell biology such as epithelial cell polarity2 , lysosomal homeostasis, and autophagy12,13.
In the last couple of years there have been a number of publications on establishing primary RPE cultures, indicating a growing interest in this area of research3,14,15. Numerous protocols for human RPE cells and non-human RPE cells such as bovine and porcine RPE cells were published16,17,18,19. However, it is more difficult to handle mouse RPE cells because of their much smaller size. Even though quite a number of publications have described protocols to isolate RPE cells from the mouse14,20,21, there are still many researchers struggling to isolate RPE cells without contamination of choroid cells or cells from neural retina debris. Here we present the protocol for establishing primary mouse RPE cell culture, including obtaining the eyes from the mouse, dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This video protocol would be especially useful for researchers who are starting to work with mouse primary RPE cultures and need guidance on the dissection techniques.

<table><tbody><tr><td>&lt;strong&gt;animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2~3-week old wildtype mice</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose</td><td>GIBCO</td><td>11965092</td><td></td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>GIBCO</td><td>15140122</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>GIBCO</td><td>11140050</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS), 1X, pH 7.4</td><td>GIBCO</td><td>10010023</td><td></td></tr><tr><td>HEPES</td><td>Cellgro</td><td>61-034</td><td></td></tr><tr><td>KOH</td><td>Sigma-Aldrich</td><td>1310-58-3</td><td></td></tr><tr><td>NaCl</td><td>Ambion</td><td>AM9759</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>GIBCO</td><td>25030-081</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Fisher Scientific</td><td>111000200</td><td></td></tr><tr><td>RPE65 antibody</td><td>A gift from Dr. Michael Redmond</td><td></td><td></td></tr><tr><td>Fluorescein Phalloidin&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>F432</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L), Alexa Flour 568&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>A11011&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Polysciences, Inc</td><td>00380</td><td></td></tr><tr><td>ZO-1 Polyclonal Antibody</td><td>ThermoFisher Scientific</td><td>40-2200</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;instruments and equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Laminar flow cabinet</td><td>Baker</td><td>SterilGARD SG403A</td><td></td></tr><tr><td>Dissecting microscope (Zoom stereomicroscope)</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>CO2 incubator with hot air sterilization</td><td>Binder</td><td>C150</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>0875712</td><td></td></tr><tr><td>12 mm Polyester Membrane Inserts Pre-Loaded in 12-Well Culture Plates, Pore Size: 0.4 &amp;micro;m, Sterile</td><td>Corning Incorporated</td><td>COR-3460</td><td></td></tr><tr><td>Westcott tenotomy scissors, std blades, sharp</td><td>STEPHENS instruments</td><td>S7-1320</td><td></td></tr><tr><td>Castroviejo suturing forceps 0.12mm</td><td>Stroz Ophthalmic Instruments</td><td>E1796</td><td></td></tr><tr><td>Crescent straight knife</td><td>Beaver-Visitec International</td><td>373808</td><td></td></tr><tr><td>Dumont Tweezers #5, 11 cm, Straight, 0.1x0.06 mm Tips, Dumostar</td><td>World Precision Instruments</td><td>500233</td><td></td></tr><tr><td>Vannas Scissors, 8 cm, 45&amp;deg; Angle, Standard</td><td>World Precision Instruments</td><td>500260</td><td></td></tr><tr><td>Millex-GS Syringe Filter Unit, 0.22 &amp;micro;m</td><td>Millipore</td><td>SLGL0250S</td><td></td></tr><tr><td>Syringe, 5 mL</td><td>BD</td><td>309632</td><td></td></tr><tr><td>Inverted Laboratory Microscope&amp;nbsp;Leica DM IL LED</td><td>&amp;nbsp;Leica&amp;nbsp;</td><td></td><td></td></tr><tr><td>Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>Barrier and non-filtered pipette tips</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

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 mouse primary RPE culture established from 2 mice in 12 mm polyester membrane insert reaches 90 - 95% confluency after 1 week in culture. After 2 weeks of culture, the cells reached 100% confluency and started to form a mosaic of hexagonal, pigmented, and bi-nucleated cells. By 3 weeks of culture, the cells continued to form the shape and pigmentation, however, after 4 weeks a portion of cells got hyperpigmented (Figure 2).
The purity of the primary RPE cell culture can be assessed by immunostaining using the RPE65 antibody (retinoid isomerohydrolase, a critical enzyme in the vertebrate visual cycle that enables the conversion of all-trans-retinyl esters to 11-cis-retinol during phototransduction) (Figure 3, red). The integrity of the cell-to-cell junctions and the presence of hexagonal shape can be demonstrated using phalloinin staining (Figure 3, green). In addition, expression of ZO-1 protein, an important component of tight junctions, can be validated using Western blotting (Figure 4) or immunostaining.
Our results demonstrate that the obtained mouse primary RPE cultures are able to proliferate, retain pigmentation, hexagonal shape and tight junctions, and express functional markers such as RPE65.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56997/56997fig1.jpg" /> 
Figure 1. Different steps of eye dissection to obtain primary RPE cell cultures. (a) After dissociation in 2% dispase, the eye is washed in growth medium. (b) The posterior eyecup is obtained by removing the cornea and lens. (c) The resulting eyecup is further cleaned by removing the associated iris pigmented epithelium. (d) After further incubation in growth medium, the eyecup is cut radially to generate quadrants. (e)The neural retina is peeled off from the remaining RPE-choroid-sclera complex. (f) The RPE sheets are gently separated from the Bruch&#39;s membrane. Note that the RPE looks more brownish (arrows), while choroid is darker and very sticky. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56997/56997fig2.jpg" /> 
Figure 2. Primary RPE cells from a 3-week old mouse cultured on a 12 mm polyester membrane insert pre-loaded in 12-well culture plates. The mouse primary RPE culture established from 2 mice&#160;in 12 mm polyester membrane insert reaches 90 - 95% confluency after 1 week in culture (a). After 2 weeks of culture, the cells reached 100% confluency and started to form a mosaic of hexagonal, pigmented and bi-nucleated cells (b). By 3 weeks of culture the cells continued to form the shape and pigmentation (c), however, after 4 weeks a portion of cells got hyperpigmented (d). Scale bar: 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56997/56997fig3.jpg" /> 
Figure 3. The RPE cell marker, RPE65, is detectable in primary mouse RPE cells. Primary mouse RPE cells after 4 weeks of culture were fixed in 4% paraformaldehyde (PFA) and incubated with either RPE65 antibody or blocking buffer as the negative control (NC). Phalloidin (green) and DAPI (blue) were also added to stain the cell membranes and nuclei. Scale bar: 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56997/56997fig4.jpg" /> 
Figure 4. The marker of tight junctions, ZO-1 is expressed in the primary RPE cells. Primary mouse RPE cells (mRPE) were harvested after 3 weeks of culture and 8 &#181;g of the cell lysate was run on a Western blot. The equivalent amount of the ARPE-19 cells (commercially available stable RPE cell line) were used as a negative control. ZO-1 was highly expressed in the primary RPE cells while being absent from the ARPE-19 cells. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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 ...

primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

Nanyang Technological University

Research

JoVE Journal

Biology

21.0K Views.  University of Pittsburgh School of Medicine. De retinale pigment epitheel (RPE) is een multi-functionele epitheel van het oog. Hier presenteren we een protocol om primaire celculturen afgeleid van de lymfkliertest RPE.

Video: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

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 ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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

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

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

Primary Culture of Porcine Retinal Pigment Epithelial Cells

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

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

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

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

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

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

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

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

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

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

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

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

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

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

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

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

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

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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