Name: primary culture of porcine retinal pigment epithelial cells
Uploaded: 2022-09-23T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

The authors would like to show their gratitude and respect to all animals contributing their cells in this study. This study was supported in part by grants from the National Key R&#38;D Program of China (2019YFA0111200, Yi Liao &#38; Yuan Gao and Grant nos. 2018YFA0107301, Wei Li). The authors thank Jingru Huang and Xiang You from Central Lab, School of Medicine, Xiamen University for technical support in confocal imaging.

The use of experimental animals complied with the regulations of the Association for Research in Vision and Ophthalmology (ARVO) and was approved by the Ethics Committee of Experimental Animal Management of Xiamen University.
1. Preparation of experimental surgical devices, tissue digestion enzyme, and cell culture buffer
<ol>
	<li>Prepare the experimental surgical devices, by cleaning and autoclaving two pairs of ophthalmic surgical scissors and forceps the day before the e...

<ol>
	<li>Tan, L. X., Germer, C. J., La Cunza, N., 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=Complement+activation,+lipid+metabolism,+and+mitochondrial+injury:+Converging+pathways+in+age-related+macular+degeneration.">Complement activation, lipid metabolism, and mitochondrial injury: Converging pathways in age-related macular degeneration.</a> Redox Biology. 37, 101781 (2020).</li><li>Caceres, P. S., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelium+polarity+in+health+and+blinding+diseases.">Retinal pigment epithelium polarity in health and blinding diseases.</a> Current Opinion in Cell Biology. 62, 37-45 (2020).</li><li>Lakkaraju, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+the+retinal+pigment+epithelium.">The cell biology of the retinal pigment epithelium.</a> Progress in Retinal and Eye Research. 78, 100846 (2020).</li><li>Somasundaran, S., Constable, I. J., Mellough, C. B., Carvalho, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelium+and+age-related+macular+degeneration:+A+review+of+major+disease+mechanisms.">Retinal pigment epithelium and age-related macular degeneration: A review of major disease mechanisms.</a> Clinical &amp; Experimental Ophthalmology. 48 (8), 1043-1056 (2020).</li><li>Ducloyer, J. B., Le Meur, G., Cronin, T., Adjali, O., Weber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+retinitis+pigmentosa.">Gene therapy for retinitis pigmentosa.</a> Medecine Sciences. 36 (6-7), 607-615 (2020).</li><li>den Hollander, A. I., Roepman, R., Koenekoop, R. K., Cremers, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leber+congenital+amaurosis:+genes,+proteins+and+disease+mechanisms.">Leber congenital amaurosis: genes, proteins and disease mechanisms.</a> Progress in Retinal and Eye Research. 27 (4), 391-419 (2008).</li><li>Samuels, I. S., Bell, B. A., Pereira, A., Saxon, J., Peachey, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+retinal+pigment+epithelium+dysfunction+is+concomitant+with+hyperglycemia+in+mouse+models+of+type+1+and+type+2+diabetes.">Early retinal pigment epithelium dysfunction is concomitant with hyperglycemia in mouse models of type 1 and type 2 diabetes.</a> Journal of Neurophysiology. 113 (4), 1085-1099 (2015).</li><li>Smith, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanin+pigment+in+the+pigmented+epithelium+of+the+retina+of+the+embryo+chick+eye+studied+in+vivo+and+in+vino.">Melanin pigment in the pigmented epithelium of the retina of the embryo chick eye studied in vivo and in vino.</a> The Anatomical Record. 18, 260-261 (1920).</li><li>Schnichels, 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=Retina+in+a+dish:+Cell+cultures,+retinal+explants+and+animal+models+for+common+diseases+of+the+retina.">Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina.</a> Progress in Retinal and Eye Research. 81, 100880 (2021).</li><li>D'Antonio-Chronowska, A., D'Antonio, M., Frazer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+iPSC-derived+retinal+pigment+epithelium+cells+(iPSC-RPE).">In vitro differentiation of human iPSC-derived retinal pigment epithelium cells (iPSC-RPE).</a> Bio-Protocol. 9 (24), 3469 (2019).</li><li>Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+of+the+human+RPE+cell+line,+ARPE-19,+induced+by+nicotinamide.">Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.</a> Experimental Eye Research. 179, 18-24 (2019).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-169 (1996).</li><li>. Scientific, T Available from: <a target="_blank" href="https://www.thermofisher.cn/document-connect/document">https://www.thermofisher.cn/document-connect/document</a> (2022)</li><li>. Toyota Available from: <a target="_blank" href="https://www.toyoboglobal.com/seihin/xr/likescience/support/manual/FSQ-201.pdf">https://www.toyoboglobal.com/seihin/xr/likescience/support/manual/FSQ-201.pdf</a> (2022)</li><li>. Abcam Available from: <a target="_blank" href="https://www.abcam.cn/protocols/immunocytochemistry-immunofluorescence-protocol">https://www.abcam.cn/protocols/immunocytochemistry-immunofluorescence-protocol</a> (2022)</li><li>. Zeiss Available from: <a target="_blank" href="https://www.zeiss.com/microscopy/en/products/software/zeiss-zen-lite.html#manuals">https://www.zeiss.com/microscopy/en/products/software/zeiss-zen-lite.html#manuals</a> (2022)</li><li>. Cell Signal Technology Available from: <a target="_blank" href="https://www.cellsignal.cn/learn-and-support/protocols/protocol-western">https://www.cellsignal.cn/learn-and-support/protocols/protocol-western</a> (2022)</li><li>Wang, 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=Reversed+senescence+of+retinal+pigment+epithelial+cell+by+coculture+with+embryonic+stem+cell+via+the+TGFbeta+and+PI3K+pathways.">Reversed senescence of retinal pigment epithelial cell by coculture with embryonic stem cell via the TGFbeta and PI3K pathways.</a> Frontiers in Cell and Developmental Biology. 8, 588050 (2020).</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> Experimental Eye Research. 126, 1-4 (2014).</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> Experimental Eye Research. 126, 5-15 (2014).</li><li>Anderson, J. M., Van Itallie, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+function+of+the+tight+junction.">Physiology and function of the tight junction.</a> Cold Spring Harbor Perspectives in Biology. 1 (2), 002584 (2009).</li><li>Nita, M., Strzalka-Mrozik, B., Grzybowski, A., Mazurek, U., Romaniuk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration+and+changes+in+the+extracellular+matrix.">Age-related macular degeneration and changes in the extracellular matrix.</a> Medical Science Monitor. 20, 1003-1016 (2014).</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> Nature Protocols. 11 (7), 1206-1218 (2016).</li><li>Langenfeld, A., Julien, S., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+isolation+and+culture+of+retinal+pigment+epithelial+cells+from+adult+rats.">An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 253 (9), 1493-1502 (2015).</li><li>Sonoda, S. <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> Nature Protocols. 4 (5), 662-673 (2009).</li></ol>

Here, a detailed and optimized protocol for the isolation, culture, and characterization of RPE cells from porcine eyeballs, which generates a good model for in vitro characterization of RPE cells and RPE-related disorder studies has been described. Methods for the isolation of the RPE from human, mouse, and rat eyes have been described previously23,24,25. However, it is difficult to obtain human eyeballs in some labora...

The retinal pigment epithelium (RPE) is located between photoreceptors and choriocapillaris in the outer layer of the retina1 with multiple functions, including forming the blood-retinal barrier, transporting and exchanging nutrients and retinal metabolites, recycling vitamin A to maintain a normal visual cycle, and phagocytosis and clearance of shed photoreceptor outer segments (POSs)2,3. Since POSs require constant self-renewal to generate vision, the RPE cells need to continuously engulf detached POSs to maintain retinal homeostasis4. Therefore, RPE dysfunction results in many blinding eye diseases, such as age-related macular degeneration (AMD)4, retinitis pigmentosa (RP)5,&#160;Leber congenital amaurosis6, diabetic retinopathy7, etc. Till now, the exact pathogenesis of most of these diseases remains elusive. As a result, RPE cell culture is established to study RPE cell biology, pathological changes, and underlying mechanisms.
As the simplest model to study cell biology, the culture of RPE cells was started as early as the 1920s8. Although ARPE-19 is widely used as RPE cells, loss of pigmentation, cobblestone morphology, and, especially, the barrier functions in this cell line raise lots of concerns9. In comparison, the culture of primary human RPE cells offers a more realistic scenario for physiological and pathological studies9. However, the relatively limited availability restricts their usage and ethical issues always exist. In addition, several groups used mouse models to culture RPE cells. However, the size of the mouse eye is small, and a single culture usually requires many mice, which is not convenient9. Recently, scientists have developed new methods to use human embryonic stem cells or induced pluripotent stem cells to derive RPE cells. Although this technique has particular potential for the treatment of inherited RPE disorders, it is time-consuming and usually requires several months to generate mature RPE cells10. To overcome these problems, here we introduce an easy-to-follow protocol to isolate and culture high-purity RPE cells in the laboratory routinely. Under suitable culture conditions, these cells can display typical RPE functions and exhibit typical RPE morphologies. Therefore, this culture method can provide a good model to understand RPE physiology, study cytotoxicity, investigate pathological mechanisms of related ocular diseases, and conduct drug screenings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARPE-19 cells</td>
			<td>CCTCC</td>
			<td>GDC0323</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Yeasen</td>
			<td>36101ES60</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM 880 with Airyscan</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch</td>
			<td>Bio-Rad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sangon</td>
			<td>F619301</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>NEST</td>
			<td>121621EH01</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>NEST</td>
			<td>29821075P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12 Medium</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td>For TER measurement</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ExCell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>0300-0108P</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Rabbit IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>5196-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>MCE</td>
			<td>HY-N0583/CS-2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution (20 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 Instrument</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Liothyronine</td>
			<td>MCE</td>
			<td>HY-A0070A/CS-4141</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM(1X)+GlutaMAX Medium</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA(100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N1</td>
			<td>Sigma-Aldrich</td>
			<td>SLCF4683</td>
			<td></td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>New Cell&#38;Molecular Biotech</td>
			<td>P10300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat Powdered Milk</td>
			<td>Solarbio</td>
			<td>D8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>SparkJade</td>
			<td>SJ-MV0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+-K+ ATPase antibody</td>
			<td>Abcam</td>
			<td>ab76020</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparation Kit(10%)</td>
			<td>Epizyme</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>primary Human RPE cells&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Generous gift from Shoubi Wang lab&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad by Dotmatics</td>
			<td>version 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktails</td>
			<td>APExBIO</td>
			<td>K1024</td>
			<td></td>
		</tr>
		<tr>
			<td>PRE65 antibody</td>
			<td>Proteintech</td>
			<td>17939-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PEDF antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-390172</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>100 x penicillin/streptomycin&#160;</td>
			<td>Biological Industries</td>
			<td>03-031-1BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>RARBIO</td>
			<td>RA-9005</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>Toyobo</td>
			<td>FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Damas-beta</td>
			<td>107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo-Fisher&#160;</td>
			<td>15596026</td>
			<td>RNA extraction solution</td>
		</tr>
		<tr>
			<td>TB Green Fast qPCR Mix</td>
			<td>Takara</td>
			<td>RR430A</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well transwell inserts</td>
			<td>Labselect</td>
			<td>14212</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF antibody</td>
			<td>Proteintech</td>
			<td>19003-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>VEGF ELISA kit</td>
			<td>Novusbio</td>
			<td>VAL106</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>ABclonal</td>
			<td>A0659</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
	</tbody>
</table>

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.

The primary porcine RPE (pRPE) cells were cultured in DMEM/Basic media with 10% FBS, and cell morphology under light microscope was photographed at 2 days (Figure 2A), 6 days (Figure 2B) and 10 days (Figure 2C) after seeding. After 1 wk, a confluent monolayer of pigmented pRPE cells with cobblestone morphologies was observed.
To better characterize the primary pRPE cells, primary human RPE cells (hRPE) at...

Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Primary Culture of Porcine Retinal Pigment Epithelial Cells

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. ...

RPE related disorders remain a major part of blending diseases, yet without effective remnants in virtual culture of primary RPE cells to serve as a good model for physiological and pathological studies of RPE related disorders. In this protocol, we provide an easy to follow protocol to culture primary IPCs, which could quickly restore and maintain later RP characteristics, we to We believe that by using this method people could quickly generate RP cells from macistate studies and the protective drug screenings which could facilitate the development of new treatment for RPE disorders. A step by step value demonstration will make this method much easier to replicate in different laboratories that want to study RP cells.To begin thaw the tissue digestion enzyme malaquad and sterilize 1X PBS. Supplemented with 2%penicillin and 2%streptomycin by filtering the solution through a 0.22 micrometer syringe filter unit. Wash the wells of the culture plate and transwell inserts with 1X PBS.Remove the PBS from the wells and transwells with a pipette and add one milliliter of fresh 1X PBS to the lower chamber and 600 microliters of 10 micrograms per milliliter Laminin solution to the upper chamber. Incubate the plates and transwell inserts in this Heliciculture Incubator overnight at 37 degrees Celsius and 5%carbon dioxide. After that, remove the laminate solution from the upper chamber and wash with one milliliter of cold 1X PBS twice before seating the cell Clean the laminate flow hood with 75%ethanol and UV light.Prepare three 50 milliliters sterile centrifuge tubes containing around 25 milliliters of 75%Ethanol, three 50 milliliters sterile centrifuge tubes containing around 25 milliliters of 1X PBS, and three 10 centimeter sterile cell culture dishes containing around 15 milliliters of 1X PBS. Soak four porcine eyeballs into about 15 milliliters of 75%Ethanol in a 10 centimeter Petri dish and use a pair of scissors and forceps to cut off all the residual connective tissues and muscles. To decontaminate the eyeballs, soak, and wash four eyeballs in three 50 milliliters sterile centrifuge tubes filled with 75%ethanol sequentially.Then wash the eyeballs in three 50 milliliters sterile centrifuge tubes filled with 1X PBS sequentially. Invert the tubes every minute to wash the eyeballs thoroughly. Move the four eyeballs into a 10 centimeter sterile cell culture dish containing 1X PBS.Trim the outer surface of each eyeball again, to remove the optic nerve and small debris. Use scissors to make a small cut at the intersection of the limbus and sclera. And then remove the cornea, iris, lens, vitreous body, and neural retina.Transfer the RPE choroid sclera complex into a new 10 centimeter cell culture dish and make four cuts to flat the eye cup into the shape of a four-leaf clover. Perform trips in EDTA solution digestion by placing the four RPE choroid sclera complexes into a new 10 centimeter dish and pouring 20 milliliters of fresh trypsin EDTA solution to merge the RPE choroid sclera complexes. Place the dish into the cell culture incubator at 37 degrees Celsius for almost 30 minutes.After 30 minutes, take the dish out of the incubator and, add 20 milliliters of pre-warm cell culture media with 10%FBS to the dish. To neutralize the trips in EDTA solution use a five milliliter transfer pipette to dissociate the RPE cells by gently pipetting several times. Collect the cell suspensions into 15 milliliters centrifuge tubes, and then gently pipette another 10 milliliters of fresh culture media with FBS to wash the RPE Choroids sclera complexes on the dish.Collect the RPE cells by centrifuge the tubes at 300 G for five minutes at room temperature. Aspirate the SuperAgent using a pipette and add another five milliliters of culture media with FBS to re-suspend the cells. Centrifuge at 300 G for another five minutes.Decant the SuperAgent and re-suspend the cells with 12 milliliters of culture media. Next seed 100, 000 to 200, 000 cells per well into 12 Well culture plates or transwell inserts. Change the culture media every two days and reduce the serum concentration to 1%when the cells fully cover the surface of the wells per insert.Change the culture media every two days until the cells are harvested. The representative images of primary porcine RPE cells on day two, day six and day 10 are shown here. The QRTPCR analysis of mRNA levels of signature genes in primary porcine RPE cells, primary human RPE cells, ARPE 19 cells and porcine RPE choroid tissue is detected by this graphical image.Here GAP DH was used as the housekeeping gene for RTPCR. Four biological replicates were used for primary porcine RPE cells and a RPE 19 cells where three biological replicates were used for primary human RPE cells and porcine RPE choroid tissue. The gene expression levels in ARPE 19 cells were set as controls.The Western blood analysis of RPE 65 proteins in ARPE 19 and porcine RPE cells and in primary human RPE cells and porcine RPE choroid tissues are shown in this figure. Vinculin was used here as a loading control. This figure shows a two week culture of porcine RPE cells in DMEM Basic media with 1%FBS.Cells cultured with or without Lamin were stained with RPE 65, sodium potassium APTAs, Z01, and Hex. TR measurements in cell sheets, cultured with or without laminin are depicted in this figure. The western blood analysis of vascular endothelial growth factor or VEGF and pigment epithelium derived factor or PEDF in primary porcine RPE and ARPE 19 cells are shown in this figure.The sectorial functions of cultured primary porcine RPE and ARPE 19 cells are presented in these images. The great part of this protocol is to dissolve RP cells from RPE chide glial complexes. We recommend using fresh trips and solution each time and the properly controlling the digestion time, to dissolve eight RP cells.

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

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, ...

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

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

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 ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

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 ...

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 ...

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

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

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 ...

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

Isolation and Culture of Primary Human Mammary Epithelial Cells

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...