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

Die Autoren möchten allen Tieren, die ihre Zellen in diese Studie einbringen, ihre Dankbarkeit und ihren Respekt entgegenbringen. Diese Studie wurde zum Teil durch Zuschüsse des National Key R&D Program of China (2019YFA0111200, Yi Liao & Yuan Gao und Grant No. 2018YFA0107301, Wei Li) unterstützt. Die Autoren danken Jingru Huang und Xiang You vom Central Lab, School of Medicine, Xiamen University für die technische Unterstützung bei der konfokalen Bildgebung.

Die Verwendung von Versuchstieren entsprach den Vorschriften der Association for Research in Vision and Ophthalmology (ARVO) und wurde von der Ethikkommission für experimentelles Tiermanagement der Universität Xiamen genehmigt.
1. Herstellung von experimentellen chirurgischen Geräten, Gewebeverdauungsenzym und Zellkulturpuffer
<ol><li>Bereiten Sie die experimentellen chirurgischen Geräte vor, indem Sie am Tag vor der Augapfeldissektion zwei Paar ophthalmochirurgische Sch...

<ol>
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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. 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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>. 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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>

Hier wurde ein detailliertes und optimiertes Protokoll für die Isolierung, Kultur und Charakterisierung von RPE-Zellen aus Augäpfeln von Schweinen beschrieben, das ein gutes Modell für die In-vitro-Charakterisierung von RPE-Zellen und RPE-bezogene Störungsstudien erzeugt. Methoden zur Isolierung des RPE aus den Augen von Menschen, Mäusen und Ratten wurden bereitsbeschrieben 23,24,25. In einigen Laboratorien ist es ...

Das retinale Pigmentepithel (RPE) befindet sich zwischen Photorezeptoren und Choriokapillaris in der äußeren Schicht der Netzhaut1 und hat mehrere Funktionen, einschließlich der Bildung der Blut-Retina-Schranke, des Transports und Austauschs von Nährstoffen und retinalen Metaboliten, des Recyclings von Vitamin A zur Aufrechterhaltung eines normalen Sehzyklus sowie der Phagozytose und Beseitigung der abgestoßenen äußeren Photorezeptorsegmente (POSs)2,3 . Da POSs eine ständige Selbsterneuerung erfordern, um das Sehen zu erzeugen, müssen die RPE-Zellen kontinuierlich abgelöste POSs verschlingen, um die retinale Homöostaseaufrechtzuerhalten 4. Daher führt eine RPE-Dysfunktion zu vielen erblindenden Augenerkrankungen, wie z.B. altersbedingte Makuladegeneration (AMD)4, Retinitis pigmentosa (RP)5, Lebersche kongenitale Amaurose6, diabetische Retinopathie7 usw. Bis heute ist die genaue Pathogenese der meisten dieser Krankheiten schwer fassbar. Infolgedessen wird die RPE-Zellkultur etabliert, um die RPE-Zellbiologie, pathologische Veränderungen und zugrunde liegende Mechanismen zu untersuchen.
Als einfachstes Modell zur Untersuchung der Zellbiologie wurde bereits in den 1920er Jahren mit der Kultivierung von RPE-Zellen begonnen8. Obwohl ARPE-19 als RPE-Zellen weit verbreitet ist, geben der Verlust der Pigmentierung, die Morphologie des Kopfsteinpflasters und insbesondere die Barrierefunktionen in dieser Zelllinie Anlass zur Sorge9. Im Vergleich dazu bietet die Kultur primärer humaner RPE-Zellen ein realistischeres Szenario für physiologische und pathologische Studien9. Die relativ begrenzte Verfügbarkeit schränkt jedoch ihre Nutzung ein und es gibt immer ethische Fragen. Darüber hinaus verwendeten mehrere Gruppen Mausmodelle, um RPE-Zellen zu kultivieren. Die Größe des Mausauges ist jedoch klein, und eine einzelne Kultur erfordert normalerweise viele Mäuse, was nicht praktisch ist9. In jüngster Zeit haben Wissenschaftler neue Methoden entwickelt, um menschliche embryonale Stammzellen oder induzierte pluripotente Stammzellen zur Gewinnung von RPE-Zellen zu verwenden. Obwohl diese Technik ein besonderes Potenzial für die Behandlung von erblichen RPE-Störungen hat, ist sie zeitaufwendig und benötigt in der Regel mehrere Monate, um reife RPE-Zellen zu erzeugen10. Um diese Probleme zu überwinden, stellen wir hier ein einfach zu befolgendes Protokoll vor, um hochreine RPE-Zellen routinemäßig im Labor zu isolieren und zu kultivieren. Unter geeigneten Kulturbedingungen können diese Zellen typische RPE-Funktionen aufweisen und typische RPE-Morphologien aufweisen. Daher kann diese Kulturmethode ein gutes Modell liefern, um die RPE-Physiologie zu verstehen, die Zytotoxizität zu untersuchen, pathologische Mechanismen verwandter Augenerkrankungen zu untersuchen und Medikamentenscreenings durchzuführen.

<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

Das retinale Pigmentepithel (RPE) ist eine Monoschicht aus polarisierten pigmentierten Epithelzellen, die sich zwischen Aderhaut und Neuroretina in der Netzhaut befindet. Mehrere Funktionen, einschließlich Phagozytose, Nährstoff- / Metabolitentransport, Vitamin-A-Stoffwechsel usw., werden vom RPE täglich durchgeführt. RPE-Zellen sind terminale differenzierte Epithelzellen mit geringer oder keiner Regenerationsfähigkeit. Der Verlust von RPE-Zellen führt zu mehreren Augenerkrankungen, die zu Sehstörungen führen, wie z. B. altersbedingte Makuladegeneration. Daher ist die Etablierung eines In-vitro-Kulturmodells von primären RPE-Zellen, das dem RPE in vivo ähnlicher ist als Zelllinien, entscheidend für die charakteristischen und mechanistischen Untersuchungen von RPE-Zellen. In Anbetracht der Tatsache, dass die Quelle menschlicher Augäpfel begrenzt ist, erstellen wir ein Protokoll zur Kultivierung primärer porciner RPE-Zellen. Durch die Verwendung dieses Protokolls können RPE-Zellen leicht von erwachsenen Augäpfeln von Schweinen getrennt werden. Anschließend heften sich diese dissoziierten Zellen an Kulturschalen / -inserts, vermehren sich zu einer konfluenten Monoschicht und stellen die Schlüsselmerkmale des Epithelgewebes in vivo innerhalb von 2 Wochen schnell wieder her. Mittels qRT-PCR wird gezeigt, dass primäre porcine RPE-Zellen mehrere Signaturgene in vergleichbaren Konzentrationen mit nativem RPE-Gewebe exprimieren, während die Expression der meisten dieser Gene in menschlichen RPE-ähnlichen Zellen, ARPE-19, verloren geht bzw. stark reduziert ist. Darüber hinaus zeigt die Immunfluoreszenzfärbung die Verteilung von Tight Junction-, Gewebepolaritäts- und Zytoskelettproteinen sowie das Vorhandensein von RPE65, einer Isomerase, die für den Vitamin-A-Stoffwechsel entscheidend ist, in kultivierten Primärzellen. Insgesamt haben wir einen einfach zu befolgenden Ansatz entwickelt, um primäre porcine RPE-Zellen mit hoher Reinheit und nativen RPE-Eigenschaften zu kultivieren, der als gutes Modell dienen könnte, um die RPE-Physiologie zu verstehen, Zelltoxizitäten zu untersuchen und Medikamentenscreenings zu erleichtern.

Die primären porcinen RPE-Zellen (pRPE) wurden in DMEM/Basismedien mit 10% FBS kultiviert, und die Zellmorphologie unter dem Lichtmikroskop wurde 2 Tage (Abbildung 2A), 6 Tage (Abbildung 2B) und 10 Tage (Abbildung 2C) nach der Aussaat fotografiert. Nach 1 Woche wurde eine konfluente Monoschicht von pigmentierten pRPE-Zellen mit Kopfsteinpflaster-Morphologien beobachtet.
Um die primären pRPE-Zellen besse...

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

Hier wird eine einfach zu befolgende Methode zur Kultivierung primärer retinaler Netzhaut-Pigmentepithelzellen in vitro vorgestellt.

Primärkultur von retinalen Pigmentepithelzellen des Schweins

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

RPE-assoziierte Störungen sind nach wie vor ein wichtiger Teil der Vermischung von Krankheiten, jedoch ohne wirksame Überreste in der virtuellen Kultur von primären RPE-Zellen, die als gutes Modell für physiologische und pathologische Studien von RPE-assoziierten Störungen dienen könnten. In diesem Protokoll stellen wir ein einfach zu befolgendes Protokoll zur Kultivierung primärer IPCs zur Verfügung, das spätere RP-Eigenschaften schnell wiederherstellen und aufrechterhalten könnte. Wir glauben, dass Menschen mit dieser Methode schnell RP-Zellen aus Makistat-Studien und den schützenden Medikamenten-Screenings erzeugen könnten, die die Entwicklung neuer Behandlungen für RPE-Störungen erleichtern könnten. Eine Schritt-für-Schritt-Demonstration des Wertes wird es viel einfacher machen, diese Methode in verschiedenen Laboratorien zu replizieren, die RP-Zellen untersuchen möchten.Um zu beginnen, tauen Sie das Gewebeverdauungsenzym Malaquad auf und sterilisieren Sie 1X PBS. Ergänzt mit 2 % Penicillin und 2 % Streptomycin durch Filtration der Lösung durch eine 0,22 Mikrometer Spritzenfiltereinheit. Waschen Sie die Vertiefungen der Kulturplatte und der Transwell-Einsätze mit 1X PBS.Entfernen Sie das PBS mit einer Pipette aus den Vertiefungen und Transwells und geben Sie einen Milliliter frisches 1X PBS in die untere Kammer und 600 Mikroliter 10 Mikrogramm pro Milliliter Lamininlösung in die obere Kammer. Inkubieren Sie die Platten und Transwell-Einsätze in diesem Heliciculture-Inkubator über Nacht bei 37 Grad Celsius und 5% Kohlendioxid. Danach entfernen Sie die Laminatlösung aus der oberen Kammer und waschen Sie zweimal mit einem Milliliter kaltem 1X PBS, bevor Sie die Zelle einsetzen. Reinigen Sie die Laminat-Flow-Haube mit 75% Ethanol und UV-Licht.Bereiten Sie drei sterile 50-Milliliter-Zentrifugenröhrchen mit etwa 25 Millilitern 75%igem Ethanol, drei sterile 50-Milliliter-Zentrifugenröhrchen mit etwa 25 Millilitern 1X PBS und drei 10-cm-sterile Zellkulturschalen mit etwa 15 Millilitern 1X PBS vor. Weichen Sie vier Schweineaugäpfel in etwa 15 Milliliter 75% Ethanol in einer 10 Zentimeter großen Petrischale ein und schneiden Sie mit einer Schere und einer Pinzette das restliche Bindegewebe und die Muskeln ab. Um die Augäpfel zu dekontaminieren, tränken und waschen Sie vier Augäpfel in drei sterilen 50-Milliliter-Zentrifugenröhrchen, die nacheinander mit 75% Ethanol gefüllt sind.Waschen Sie dann die Augäpfel nacheinander in drei sterilen 50-Milliliter-Zentrifugenröhrchen, die mit 1X PBS gefüllt sind. Drehen Sie die Schläuche jede Minute um, um die Augäpfel gründlich zu waschen. Bewegen Sie die vier Augäpfel in eine 10 Zentimeter große sterile Zellkulturschale mit 1X PBS.Schneiden Sie die äußere Oberfläche jedes Augapfels erneut ab, um den Sehnerv und kleine Trümmer zu entfernen. Verwenden Sie eine Schere, um einen kleinen Schnitt am Schnittpunkt von Limbus und Sklera zu machen. Und dann entfernen Sie die Hornhaut, die Iris, die Linse, den Glaskörper und die neurale Netzhaut.Übertragen Sie den RPE-Aderhaut-Sklera-Komplex in eine neue 10-Zentimeter-Zellkulturschale und machen Sie vier Schnitte, um die Augenmuschel in die Form eines vierblättrigen Kleeblatts zu glätten. Führen Sie Ausflüge in die Verdauung von EDTA-Lösungen durch, indem Sie die vier RPE-Aderhaut-Sklera-Komplexe in eine neue 10-Zentimeter-Schale geben und 20 Milliliter frische Trypsin-EDTA-Lösung gießen, um die RPE-Aderhaut-Sklera-Komplexe zu verschmelzen. Stellen Sie die Schale für fast 30 Minuten in den Zellkultur-Inkubator bei 37 Grad Celsius.Nach 30 Minuten nehmen Sie die Schale aus dem Inkubator und geben Sie 20 Milliliter vorwarme Zellkulturmedien mit 10% FBS in die Schale. Um die Trips in der EDTA-Lösung zu neutralisieren, verwenden Sie eine Fünf-Milliliter-Transferpipette, um die RPE-Zellen durch vorsichtiges Pipettieren mehrmals zu dissoziieren. Sammeln Sie die Zellsuspensionen in 15-Milliliter-Zentrifugenröhrchen und pipettieren Sie dann vorsichtig weitere 10 Milliliter frisches Nährmedium mit FBS, um die RPE-Aderhaut-Sklera-Komplexe auf der Schale zu waschen.Sammeln Sie die RPE-Zellen, indem Sie die Röhrchen bei 300 g für fünf Minuten bei Raumtemperatur zentrifugieren. Saugen Sie den SuperAgent mit einer Pipette ab und fügen Sie weitere fünf Milliliter Nährmedien mit FBS hinzu, um die Zellen wieder zu suspendieren. Zentrifuge bei 300 g für weitere fünf Minuten.Dekantieren Sie den SuperAgent und suspendieren Sie die Zellen mit 12 Milliliter Nährmedien. Als nächstes werden 100.000 bis 200.000 Zellen pro Vertiefung in 12 Well-Kulturplatten oder Transwell-Einsätze gesät. Wechseln Sie die Nährmedien alle zwei Tage und reduzieren Sie die Serumkonzentration auf 1%, wenn die Zellen die Oberfläche der Vertiefungen pro Einsatz vollständig bedecken.Wechseln Sie die Nährmedien alle zwei Tage, bis die Zellen geerntet sind. Die repräsentativen Bilder der primären porcinen RPE-Zellen am zweiten, sechsten und 10. Tag sind hier zu sehen. Die QRTPCR-Analyse der mRNA-Spiegel von Signaturgenen in primären porcinen RPE-Zellen, primären humanen RPE-Zellen, ARPE-19-Zellen und porcinem RPE-Aderhautgewebe wird durch dieses grafische Bild detektiert.Hier wurde GAP DH als Housekeeping-Gen für RTPCR verwendet. Vier biologische Replikate wurden für primäre porcine RPE-Zellen und eine RPE 19-Zelle verwendet, während drei biologische Replikate für primäre humane RPE-Zellen und porcines RPE-Aderhautgewebe verwendet wurden. Die Genexpressionsniveaus in ARPE-19-Zellen wurden als Kontrollen festgelegt.Die westliche Blutanalyse von RPE-65-Proteinen in ARPE-19- und porcinen RPE-Zellen sowie in primären humanen RPE-Zellen und porcinen RPE-Aderhautgeweben ist in dieser Abbildung dargestellt. Vinculin wurde hier als Belastungskontrolle eingesetzt. Diese Abbildung zeigt eine zweiwöchige Kultur von porcinen RPE-Zellen in DMEM-Basic-Medien mit 1%FBS.Zellen, die mit oder ohne Lamin kultiviert wurden, wurden mit RPE 65, Natrium-Kalium-APTAs, Z01 und Hex gefärbt. TR-Messungen in Zellblättern, die mit oder ohne Laminin kultiviert wurden, sind in dieser Abbildung dargestellt. Die westliche Blutanalyse des vaskulären endothelialen Wachstumsfaktors (VEGF) und des Pigmentepithel-abgeleiteten Faktors (PEDF) in primären porcinen RPE- und ARPE-19-Zellen ist in dieser Abbildung dargestellt.In diesen Bildern werden die sektoralen Funktionen von kultivierten primären porcinen RPE- und ARPE-19-Zellen dargestellt. Der größte Teil dieses Protokolls besteht darin, RP-Zellen aus RPE-Chide-Glia-Komplexen aufzulösen. Wir empfehlen, jedes Mal frische Trips und Lösungen zu verwenden und die Verdauungszeit richtig zu kontrollieren, um acht RP-Zellen aufzulösen.

primary-culture-of-porcine-retinal-pigment-epithelial-cells

Research

JoVE Journal

Biology

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 uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex vivo-Kultur von primären humanen Eileiter Epithelzellen

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 ...

Forschung

Biologie

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.

MicroRNA Expressionsprofile der Menschen iPS-Zellen, Retinapigmentepithel Abgeleitet aus iPS und Fetal Retinapigmentepithel

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

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

Subretinale Transplantation von humanen embryonalen Stammzellen abgeleitet retinalen Pigment Epithelzellen zu einem großen Augen geographische Atrophie

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

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Primären Zellkulturen aus das retinale Pigmentepithel Maus

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

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

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Isolierung, Kultur und Gentechnik von primären Pigmentepithelzellen von Säugetieren für die nicht-virale Gentherapie

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 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.

Effiziente Dissektion und Kultur der primären Maus retinalen Pigmentepithelzellen

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 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.

Isolierung von primären retinalen pigmentierten Epithelzellen der Maus

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

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

Echtzeitanalyse der Bioenergetik in primären humanen retinalen Pigmentepithelzellen mittels hochauflösender Respirometrie

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

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

Isolierung von retinalen Pigmentepithelzellen aus Meerschweinchenaugen

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

Protokoll zur Isolierung von Brustepithelzellen aus menschlichem Gewebe

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Autor im Rampenlicht: Integration östlicher und westlicher Medizin zur Behandlung von granulomatöser Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Isolierung und Kultivierung primärer humaner Brustepithelzellen

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