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

Os autores gostariam de mostrar sua gratidão e respeito a todos os animais que contribuem com suas células neste estudo. Este estudo foi apoiado em parte por doações do Programa Nacional de P & D da China (2019YFA0111200, Yi Liao & Yuan Gao e Grant nos. 2018YFA0107301, Wei Li). Os autores agradecem a Jingru Huang e Xiang You do Laboratório Central, Escola de Medicina, Universidade de Xiamen, pelo apoio técnico em imagens confocais.

O uso de animais experimentais cumpriu os regulamentos da Associação de Pesquisa em Visão e Oftalmologia (ARVO) e foi aprovado pelo Comitê de Ética de Manejo de Animais Experimentais da Universidade de Xiamen.
1. Preparação de dispositivos cirúrgicos experimentais, enzima de digestão tecidual e tampão de cultura celular
<ol><li>Preparar os dispositivos cirúrgicos experimentais, limpando e autoclavando dois pares de tesouras cirúrgicas oftálmicas e fórceps no di...

<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>

Aqui, um protocolo detalhado e otimizado para o isolamento, cultura e caracterização de células RPE de globos oculares suínos, o que gera um bom modelo para caracterização in vitro de células RPE e estudos de distúrbios relacionados ao RPE foi descrito. Métodos para o isolamento do PSE de olhos humanos, camundongos e ratos foram descritos anteriormente23,24,25. No entanto, é difícil obter globos oculares huma...

O epitélio pigmentar da retina (EPR) está localizado entre fotorreceptores e coriocapilares na camada externa da retina1 com múltiplas funções, incluindo a formação da barreira sangue-retina, transporte e troca de nutrientes e metabólitos da retina, reciclagem de vitamina A para manter um ciclo visual normal e fagocitose e depuração dos segmentos externos fotorreceptores (POSs)2,3 . Como os POSs requerem auto-renovação constante para gerar visão, as células RPE precisam engolir continuamente os POSs destacados para manter a homeostase da retina4. Portanto, a disfunção do PSE resulta em muitas doenças oculares cegantes, como degeneração macular relacionada à idade (DMRI)4, retinite pigmentosa (RP)5, amaurose congênita de Leber6, retinopatia diabética7, etc. Até agora, a patogênese exata da maioria dessas doenças permanece indescritível. Como resultado, a cultura de células RPE é estabelecida para estudar a biologia celular RPE, alterações patológicas e mecanismos subjacentes.
Como o modelo mais simples para estudar a biologia celular, a cultura de células RPE foi iniciada já na década de 19208. Embora o ARPE-19 seja amplamente utilizado como células RPE, a perda de pigmentação, a morfologia do paralelepípedo e, especialmente, as funções de barreira nessa linhagem celular levantam muitas preocupações9. Em comparação, a cultura de células primárias de EPR humanas oferece um cenário mais realista para estudos fisiológicos e patológicos9. No entanto, a disponibilidade relativamente limitada restringe seu uso e sempre existem questões éticas. Além disso, vários grupos usaram modelos de camundongos para cultivar células RPE. No entanto, o tamanho do olho do rato é pequeno, e uma única cultura geralmente requer muitos ratos, o que não é conveniente9. Recentemente, os cientistas desenvolveram novos métodos para usar células-tronco embrionárias humanas ou células-tronco pluripotentes induzidas para derivar células RPE. Embora essa técnica tenha um potencial particular para o tratamento de distúrbios hereditários do PSE, é demorada e geralmente requer vários meses para gerar células maduras do PSE10. Para superar esses problemas, aqui apresentamos um protocolo fácil de seguir para isolar e cultivar células RPE de alta pureza em laboratório rotineiramente. Sob condições de cultura adequadas, essas células podem exibir funções RPE típicas e exibir morfologias RPE típicas. Portanto, esse método de cultura pode fornecer um bom modelo para entender a fisiologia do EPR, estudar a citotoxicidade, investigar mecanismos patológicos de doenças oculares relacionadas e realizar exames de drogas.

<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

O epitélio pigmentar da retina (EPR) é uma monocamada de células epiteliais pigmentadas polarizadas, localizadas entre a coroide e a neurorretina na retina. Múltiplas funções, incluindo fagocitose, transporte de nutrientes / metabólitos, metabolismo da vitamina A, etc., são conduzidas pelo PSE diariamente. As células PSE são células epiteliais terminalmente diferenciadas com pouca ou nenhuma capacidade regenerativa. A perda de células PSE resulta em múltiplas doenças oculares que levam à deficiência visual, como a degeneração macular relacionada à idade. Portanto, o estabelecimento de um modelo de cultura in vitro de células primárias de EPR, que mais se assemelha ao PSE in vivo do que linhagens celulares, é fundamental para os estudos característicos e mecanicistas de células de EPR. Considerando o fato de que a fonte de globos oculares humanos é limitada, criamos um protocolo para cultivar células primárias de PSE suína. Usando este protocolo, as células RPE podem ser facilmente dissociadas dos globos oculares suínos adultos. Posteriormente, essas células dissociadas se ligam a placas / inserções de cultura, proliferam para formar uma monocamada confluente e restabelecem rapidamente as principais características do tecido epitelial in vivo dentro de 2 semanas. Por qRT-PCR, demonstra-se que as células primárias de PSE suína expressam múltiplos genes de assinatura em níveis comparáveis com o tecido RPE nativo, enquanto as expressões da maioria desses genes são perdidas/altamente reduzidas em células humanas semelhantes a RPE, ARPE-19. Além disso, a coloração por imunofluorescência mostra a distribuição de proteínas de junção apertada, polaridade tecidual e citoesqueleto, bem como a presença de RPE65, uma isomerase crítica para o metabolismo da vitamina A, em células primárias cultivadas. No total, desenvolvemos uma abordagem fácil de seguir para cultivar células primárias de PSE suíno com alta pureza e características nativas de RPE, o que poderia servir como um bom modelo para entender a fisiologia da EPR, estudar toxicidades celulares e facilitar exames de drogas.

As células primárias de PSE suíno (pRPE) foram cultivadas em meio DMEM/Basic com 10% de FBS, e a morfologia celular em microscópio de luz foi fotografada aos 2 dias (Figura 2A), 6 dias (Figura 2B) e 10 dias (Figura 2C) após a semeadura. Após 1 semana, observou-se uma monocamada confluente de células de pRPE pigmentadas com morfologias de paralelepípedos.
Para melhor caracterizar as células primá...

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

Aqui, um método fácil de seguir para a cultura de células epiteliais primárias do pigmento da retina suína in vitro é apresentado.

Cultura Primária de Células Epiteliais do Pigmento da Retina Suína

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

Os distúrbios relacionados ao PSE continuam sendo uma parte importante das doenças de mistura, mas sem remanescentes efetivos na cultura virtual de células primárias de EPR para servir como um bom modelo para estudos fisiológicos e patológicos de distúrbios relacionados ao PSE. Neste protocolo, fornecemos um protocolo fácil de seguir para a cultura de IPCs primárias, que poderiam restaurar e manter rapidamente as características de RP posteriores, acreditamos que, usando este método, as pessoas poderiam gerar rapidamente células RP a partir de estudos de macistate e os rastreios de drogas protetoras que poderiam facilitar o desenvolvimento de novo tratamento para distúrbios de RPE. Uma demonstração de valor passo a passo tornará este método muito mais fácil de replicar em diferentes laboratórios que desejam estudar células RP.Para começar a descongelar a enzima de digestão tecidual malaquad e esterilizar 1X PBS. Suplementado com 2% de penicilina e 2% de estreptomicina filtrando a solução através de uma unidade de filtro de seringa de 0,22 micrômetros. Lave os poços da placa de cultura e as inserções transwell com PBS 1X.Retire o PBS dos poços e transwells com uma pipeta e adicione um mililitro de PBS 1X fresco à câmara inferior e 600 microlitros de 10 microgramas por mililitro de solução de laminina à câmara superior. Incubar as placas e as inserções transwell nesta Incubadora de Helicicultura durante a noite a 37 graus Celsius e 5% de dióxido de carbono. Depois disso, remova a solução laminada da câmara superior e lave com um mililitro de PBS 1X frio duas vezes antes de sentar a célula Limpe o exaustor de fluxo laminado com etanol a 75% e luz UV.Prepare três tubos de centrífuga estéreis de 50 mililitros contendo cerca de 25 mililitros de etanol a 75%, três tubos de centrífuga estéreis de 50 mililitros contendo cerca de 25 mililitros de PBS 1X e três pratos de cultura de células estéreis de 10 centímetros contendo cerca de 15 mililitros de PBS 1X. Mergulhe quatro globos oculares suínos em cerca de 15 mililitros de etanol a 75% em uma placa de Petri de 10 centímetros e use uma tesoura e pinça para cortar todos os tecidos conjuntivos e músculos residuais. Para descontaminar os globos oculares, mergulhe e lave quatro globos oculares em três tubos de centrífuga estéreis de 50 mililitros preenchidos com etanol a 75% sequencialmente.Em seguida, lave os globos oculares em três tubos de centrífuga estéreis de 50 mililitros preenchidos com 1X PBS sequencialmente. Inverta os tubos a cada minuto para lavar bem os globos oculares. Mova os quatro globos oculares para um prato de cultura de células estéreis de 10 centímetros contendo 1X PBS.Apare a superfície externa de cada globo ocular novamente, para remover o nervo óptico e pequenos detritos. Use uma tesoura para fazer um pequeno corte na interseção do limbo e da esclera. E, em seguida, remova a córnea, íris, lente, corpo vítreo e retina neural.Transfira o complexo da esclera coroide RPE para um novo prato de cultura celular de 10 centímetros e faça quatro cortes para achatar o copo do olho na forma de um trevo de quatro folhas. Realize viagens na digestão de solução de EDTA colocando os quatro complexos de esclera coroide RPE em um novo prato de 10 centímetros e despejando 20 mililitros de solução EDTA de tripsina fresca para mesclar os complexos de esclera coroide de RPE. Coloque o prato na incubadora de cultura de células a 37 graus Celsius por quase 30 minutos.Após 30 minutos, retire o prato da incubadora e, adicione 20 mililitros de meio de cultura celular pré-quente com 10% FBS ao prato. Para neutralizar as viagens em solução de EDTA, use uma pipeta de transferência de cinco mililitros para dissociar as células RPE pipetando suavemente várias vezes. Colete as suspensões celulares em tubos de centrífuga de 15 mililitros e, em seguida, pipete suavemente outros 10 mililitros de meios de cultura frescos com FBS para lavar os complexos de esclera de coroides RPE no prato.Recolher as células RPE centrifugando os tubos a 300 G durante cinco minutos à temperatura ambiente. Aspirar o SuperAgente usando uma pipeta e adicionar mais cinco mililitros de meios de cultura com FBS para suspender novamente as células. Centrífuga a 300 G por mais cinco minutos.Decantar o SuperAgente e ressuspender as células com 12 mililitros de meios de cultura. Próxima semente 100, 000 a 200, 000 células por poço em 12 placas de cultura de poço ou inserções transwell. Mudar o meio de cultura a cada dois dias e reduzir a concentração sérica para 1% quando as células cobrirem totalmente a superfície dos poços por inserção.Troque o meio de cultura a cada dois dias até que as células sejam colhidas. As imagens representativas das células primárias de PSE suína no segundo dia, no sexto dia e no dia 10 são mostradas aqui. A análise QRTPCR dos níveis de mRNA de genes de assinatura em células primárias de PSE suíno, células primárias de RPE humana, células ARPE 19 e tecido coroide de RPE suíno é detectada por esta imagem gráfica.Aqui, o GAP DH foi usado como o gene de limpeza para RTPCR. Quatro replicações biológicas foram usadas para células primárias de RPE suína e uma célula RPE 19, onde três replicações biológicas foram usadas para células primárias de RPE humana e tecido coroide de RPE suíno. Os níveis de expressão gênica nas células ARPE 19 foram definidos como controles.A análise sanguínea ocidental das proteínas RPE 65 em células ARPE 19 e RPE suína e em células primárias de RPE humana e tecidos coroides de RPE suíno são mostradas nesta figura. Vinculin foi usado aqui como um controle de carga. Esta figura mostra uma cultura de duas semanas de células RPE suínas em meio DMEM Basic com 1% de FBS.As células cultivadas com ou sem Lamin foram coradas com RPE 65, APTAs de sódio e potássio, Z01 e Hex. As medidas de TR em folhas celulares, cultivadas com ou sem laminina são representadas nesta figura. A análise sanguínea ocidental do fator de crescimento endotelial vascular ou VEGF e fator derivado do epitélio pigmentar ou PEDF em células primárias de PSE suíno e ARPE 19 são mostradas nesta figura.As funções setoriais das células PSE primária suína cultivada e ARPE 19 são apresentadas nestas imagens. A grande parte deste protocolo é dissolver as células RP dos complexos gliais de chide RPE. Recomendamos o uso de viagens frescas e solução a cada vez e o controle adequado do tempo de digestão, para dissolver oito células RP.

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 de Cultura Primária células epiteliais trompa de Falópio

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 ...

Biologia

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.

Perfis de expressão de microRNA iPS Human Cells, Retinal Pigment Epithelium Derivado de Ips, e 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, 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.

Subretinal transplante de células epiteliais de pigmento retinal derivado de células-tronco embrionárias humanas em um modelo de grandes olhos de atrofia geográfica

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.

Culturas de células primárias do Mouse epithelium Retinal do Pigment

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.

Isolamento, Cultura e Engenharia Genética de Células Epiteliais de Pigmento Primário mamífero para terapia genética não viral

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.

Dissecção eficiente e cultura de células epiteliais de pigmento de retina do rato primário

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.

Isolamento das células epitélio pigmentadas do rato primário

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.

Análise em Tempo Real da Bioenergética em Células Epiteliais Pigmentares Pigmentares Humanas Primárias Usando Respirometria de Alta Resolução

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.

Isolamento de Células Epiteliais Pigmentares da Retina de Olhos de Cobaia

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

Protocolo para Isolar Células Epiteliais Mamárias de Tecido Humano

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 em destaque: Integrando a medicina oriental e ocidental para o tratamento da mastite granulomatosa

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.

Isolamento e Cultura de Células Epiteliais Mamárias Humanas Primárias

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