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 border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">animals</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">2~3-week old wildtype mice</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:123px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">reagents</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), high glucose</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">11965092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dispase II</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:123px;">D4693</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:123px;">F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Phosphate-buffered saline (PBS), 1X, pH 7.4</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">HEPES</td>
			<td style="width:123px;">Cellgro</td>
			<td style="width:123px;">61-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">KOH</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">1310-58-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaCl</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:123px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">L-Glutamine (200 mM)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ethyl Alcohol</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:123px;">111000200</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:38px;width:347px;">RPE65 antibody</td>
			<td style="width:123px;">A gift from Dr. Michael Redmond</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fluorescein Phalloidin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">F432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Goat anti-Rabbit IgG (H+L), Alexa Flour 568&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">A11011&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Goat serum</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DAPI</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Paraformaldehyde (PFA)</td>
			<td style="width:123px;">Polysciences, Inc</td>
			<td style="width:123px;">00380</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:38px;width:347px;">ZO-1 Polyclonal Antibody</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:123px;">40-2200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:123px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">instruments and equipments</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Laminar flow cabinet</td>
			<td style="width:123px;">Baker</td>
			<td style="width:123px;">SterilGARD SG403A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dissecting microscope (Zoom stereomicroscope)</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:123px;">SMZ1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">CO2 incubator with hot air sterilization</td>
			<td style="width:123px;">Binder</td>
			<td style="width:123px;">C150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:123px;">5702</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Petri dishes</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:123px;">0875712</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">12 mm Polyester Membrane Inserts Pre-Loaded in 12-Well Culture Plates, Pore Size: 0.4 &#181;m, Sterile</td>
			<td style="width:123px;">Corning Incorporated</td>
			<td style="width:123px;">COR-3460</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Westcott tenotomy scissors, std blades, sharp</td>
			<td style="width:123px;">STEPHENS instruments</td>
			<td style="width:123px;">S7-1320</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Castroviejo suturing forceps 0.12mm</td>
			<td style="width:123px;">Stroz Ophthalmic Instruments</td>
			<td style="width:123px;">E1796</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Crescent straight knife</td>
			<td style="width:123px;">Beaver-Visitec International</td>
			<td style="width:123px;">373808</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Dumont Tweezers #5, 11 cm, Straight, 0.1x0.06 mm Tips, Dumostar</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">500233</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Vannas Scissors, 8 cm, 45&#176; Angle, Standard</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">500260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Millex-GS Syringe Filter Unit, 0.22 &#181;m</td>
			<td style="width:123px;">Millipore</td>
			<td style="width:123px;">SLGL0250S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Syringe, 5 mL</td>
			<td style="width:123px;">BD</td>
			<td style="width:123px;">309632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Inverted Laboratory Microscope&#160;Leica DM IL LED</td>
			<td style="width:123px;">&#160;Leica&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipette</td>
			<td style="width:123px;">Gilson</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Barrier and non-filtered pipette tips</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:123px;"></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

20.9K Views.  University of Pittsburgh School of Medicine. 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.

Video: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

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 ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

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 ...