Name: primary cell cultures from the mouse retinal pigment epithelium
Uploaded: 2018-03-16T13:30:00
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 so...

<ol>
	<li>Mart&#237;nez-Morales, J. R., Rodrigo, I., Bovolenta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+development:+a+view+from+the+retina+pigmented+epithelium.">Eye development: a view from the retina pigmented epithelium.</a> Bioessays. 26, 766-777 (2004).</li><li>Lehmann, G. L., Benedicto, I., Philp, N. J., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+protein+polarity+and+trafficking+in+RPE+cells:+past,+present+and+future.">Plasma membrane protein polarity and trafficking in RPE cells: past, present and future.</a> Exp Eye Res. 126, 5-15 (2014).</li><li>Fronk, A. H., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+retinal+pigment+epithelial+cells:+a+review+of+current+protocols+and+future+recommendations.">Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.</a> J Tissue Eng. 7, (2016).</li><li>Rizzolo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+properties+of+cultured+retinal+pigment+epithelium.">Barrier properties of cultured retinal pigment epithelium.</a> Exp Eye Res. 126, 16-26 (2014).</li><li>Lu, F., Yan, D., Zhou, X., Hu, D. N., Qu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+melanin-related+genes+in+cultured+adult+human+retinal+pigment+epithelium+and+uveal+melanoma+cells.">Expression of melanin-related genes in cultured adult human retinal pigment epithelium and uveal melanoma cells.</a> Mol Vis. 13, 2066-2072 (2007).</li><li>Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+melanin+and+lipofuscin+in+RPE+cell+culture+models.">Studying melanin and lipofuscin in RPE cell culture models.</a> Exp Eye Res. 126, 61-67 (2014).</li><li>Saini, J. S., Temple, S., Stern, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Retinal+Pigment+Epithelium+Stem+Cell+(RPESC).">Human Retinal Pigment Epithelium Stem Cell (RPESC).</a> Adv Exp Med Biol. 854, 557-562 (2016).</li><li>Akrami, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelium+culture;a+potential+source+of+retinal+stem+cells.">Retinal pigment epithelium culture;a potential source of retinal stem cells.</a> J Ophthalmic Vis Res. 4, 134-141 (2009).</li><li>Hu, J., Bok, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cultured+human+fetal+retinal+pigment+epithelium+in+studies+of+the+classical+retinoid+visual+cycle+and+retinoid-based+disease+processes.">The use of cultured human fetal retinal pigment epithelium in studies of the classical retinoid visual cycle and retinoid-based disease processes.</a> Exp Eye Res. , 46-50 (2014).</li><li>Mazzoni, F., Safa, H., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+photoreceptor+outer+segment+phagocytosis:+use+and+utility+of+RPE+cells+in+culture.">Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.</a> Exp Eye Res. 126, 51-60 (2014).</li><li>Reichhart, N., Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+and+transporters+of+the+retinal+pigment+epithelium.">Ion channels and transporters of the retinal pigment epithelium.</a> Exp Eye Res. 126, 27-37 (2014).</li><li>Valapala, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal-mediated+waste+clearance+in+retinal+pigment+epithelial+cells+is+regulated+by+CRYBA1/&#946;A3/A1-crystallin+via+V-ATPase-MTORC1+signaling.">Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/&#946;A3/A1-crystallin via V-ATPase-MTORC1 signaling.</a> Autophagy. 10, 480-496 (2014).</li><li>Shang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amino+acid+transporter+SLC36A4+regulates+the+amino+acid+pool+in+retinal+pigmented+epithelial+cells+and+mediates+the+mechanistic+target+of+rapamycin,+complex+1+signaling.">The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.</a> Aging Cell. , (2017).</li><li>Fernandez-Godino, R., Garland, D. L., Pierce, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+characterization+of+primary+mouse+RPE+cells.">Isolation, culture and characterization of primary mouse RPE cells.</a> Nat Protoc. 11, 1206-1218 (2016).</li><li>Pfeffer, B. A., Philp, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+retinal+pigment+epithelium:+Special+Issue.">Cell culture of retinal pigment epithelium: Special Issue.</a> Exp Eye Res. 126, 1-4 (2014).</li><li>Singh, S., Woerly, S., McLaughlin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+artificial+substrates+for+retinal+pigment+epithelial+monolayer+transplantation.">Natural and artificial substrates for retinal pigment epithelial monolayer transplantation.</a> Biomaterials. 22, 3337-3343 (2001).</li><li>Sonoda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+the+culture+and+differentiation+of+highly+polarized+human+retinal+pigment+epithelial+cells.">A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.</a> Nat Protoc. 4, 662-673 (2009).</li><li>Ho, T. C., Del Priore, L. V., Kaplan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+of+retinal+pigment+epithelium+following+isolation+with+a+gelatin+matrix+technique.">Tissue culture of retinal pigment epithelium following isolation with a gelatin matrix technique.</a> Experimental eye research. 64, 133-139 (1997).</li><li>Toops, K. A., Tan, L. X., Lakkaraju, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+three-step+protocol+for+live+imaging+of+intracellular+traffic+in+polarized+primary+porcine+RPE+monolayers.">A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers.</a> Exp Eye Res. , 74-85 (2014).</li><li>Gibbs, D., Kitamoto, J., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+phagocytosis+by+retinal+pigmented+epithelium+that+lacks+myosin+VIIa,+the+Usher+syndrome+1B+protein.">Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein.</a> Proc Natl Acad Sci USA. 100, 6481-6486 (2003).</li><li>Gibbs, D., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+mouse+retinal+pigmented+epithelial+cells.">Isolation and culture of primary mouse retinal pigmented epithelial cells.</a> Adv Exp Med Biol. 533, 347-352 (2003).</li><li>Pitk&#228;nen, L., Ranta, V. P., Moilanen, H., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+retinal+pigment+epithelium:+effects+of+permeant+molecular+weight+and+lipophilicity.">Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity.</a> Invest Ophthalmol Vis Sci. 46, 641-646 (2005).</li><li>Mao, Y., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+photoreceptor+outer+segment+phagocytosis+by+RPE+cells+in+culture.">Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture.</a> Methods Mol Biol. 935, 285-295 (2013).</li></ol>

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, ...

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>
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			<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>
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			<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>
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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).
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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 ...

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