Este estudo foi parcialmente financiado pela Fundação de BrightFocus (para DS).

Procedimentos envolvendo assuntos animais foram aprovados pelo Comitê de uso (IACUC) na Universidade de Pittsburgh e institucional Cuidado Animal
1. Preparar soluções
<ol><li>Prepare o crescimento médio pela completa Dulbecco modificado águia médio (DMEM), glicose alta com 10% de soro fetal bovino (FBS), 1% penicilina/estreptomicina, 2,5 mM L-glutamina e 1 x MEM não essenciais aminoácidos. Pré-aquecer a mídia na incubadora de 37 ° C antes do uso.</li>
	<li>Prepare a solução de trabalho 2% (wt/vol) dispase II:<ol>
<li>Prepare uma solução stock de dispase 100mg/mL II dissolvendo-se 30 mg de dispase II em 300 µ l HEPES salino (50mm KOH/HEPES pH 7,4, 150 mM NaCl). Este volume é para 3-4 ratos, pode ser ajustado para cima ou para baixo com base no número de ratos para a experiência). Estas ações podem ser armazenadas pelo menos 1 semana a 4 ° C.</li>
		<li>Prepare a solução de trabalho de 2% dispase, juntando 300 µ l a 100 mg/ml estoque II dispase a 1,2 mL de DMEM estéril, glicose alta e filtrar a solução através de um filtro de 0,22 µm. Esta etapa deve ser executada no fluxo laminar em condições estéreis. Pré-aqueça o preparado dispase II em incubadora 37 ° C antes do uso.</li>
	</ol>
</li>
</ol>2. obtenção dos olhos de rato
<ol><li>Eutanásia o mouse usando qualquer método aprovado e colocá-lo em um absorvente.</li>
	<li>Usando luvas, colocar o polegar e o dedo indicador ao redor dos olhos e empurre suavemente a pele para proptose do globo ocular.</li>
	<li>Inserir a ponta da tesoura angulada entre a pele e o globo ocular e corte com cuidado para fora do olho. Para evitar contaminação, brevemente, mergulhe o olho em etanol a 70%.</li>
	<li>Coloca imediatamente o olho no tampão fosfato salino (PBS) em uma placa de Petri.</li>
	<li>Repita as etapas para remover o segundo olho da 2.2-2.4.</li>
</ol>3. dissecação do olho
<ol><li>Remova cuidadosamente o tecido conjuntivo do olho no microscópio de dissecação. Isso pode ser feito em PBS ou sobre o prato de Petri a seco.<ol>
<li>Use pinça de sutura para levantar os tecidos conjuntivos do globo ocular e cortá-los com uma tesoura Vannas. Não corte a esclera porque é praticamente impossível obter as viseiras posteriores intactas se a esclera é cortada.</li>
		<li>Após a limpeza do tecido conjuntivo, mantenha os olhos resultantes em PBS para a próxima etapa de lavagem. Nota: Todas as etapas a seguir devem ser executadas no fluxo laminar em condições estéreis.</li>
	</ol>
</li>
	<li>Lave os olhos duas vezes em previamente aquecido a 37 ° C DMEM (glicose alta). Transferi com cuidado os globos oculares para o pré-aquecido 37 ° C DMEM (glicose alta) usando fórceps. Aspire os globos oculares médio e transferência para meio fresco em um prato novo.</li>
	<li>Aspire médio DMEM (glicose alta) e incube-os olhos da solução de trabalho II dispase de 2% (wt/vol) pré-aquecido por 45 min em incubadora a 37 ° C. Nota: Em todas as etapas a seguir, coloque e manipular os olhos ou viseiras no meio de crescimento, que vão proteger o tecido e fornecer um meio bom para a dissecação.</li>
	<li>Remover a solução II dispase e lavar os olhos duas vezes, no meio de crescimento por aspiração do meio velho e adicionando o novo meio de crescimento previamente aquecido a 37 ° C (Figura 1a). Nota: Após a dissociação por dispase II, os globos oculares se tornam macios.</li>
	<li>O olho com a pinça e fazer uma incisão ao redor da ora serrata de cada olho usando Tesoura Vannas sob o microscópio dissecação colocado no fluxo laminar do armário.<ol>
<li>Manter o globo ocular na solução, Retire cuidadosamente a córnea anterior estendendo o corte em torno da circunferência de serrata ora e se afastando da córnea anterior após o corte foi completo.</li>
		<li>Retire a cápsula da lente e epitélio associado íris pigmentada puxando suavemente-os fora a viseira com pinça teethed (Figura 1b, 1C).</li>
	</ol>
</li>
	<li>Incube as viseiras resultantes posteriores no meio de crescimento por 20 min a 37 ° C para facilitar a separação da retina neural o RPE.</li>
	<li>Corte as viseiras posteriores em 4 pétalas ao microscópio dissecação colocado no fluxo laminar do armário. Os cortes devem ser tempo suficiente para achatar a viseira, mas curto o suficiente para manter as pétalas conectadas (Figura 1D).<ol>
<li>Achate a viseira e remover a retina neural, muito delicadamente, puxando-o com pinça, mantendo o restante complexo RPE-coroide-esclera com outra pinça (Figura 1e). Comece a puxar das bordas, em vez do centro.</li>
	</ol>
</li>
	<li>Transferência de de que todos os tecidos esquartejados para uma nova placa de cultura estéril cheia pré aquecido médio de crescimento de 37 ° C.</li>
</ol>4. isolamento de células primárias de RPE
<ol><li>Segurando uma pétala do complexo coroide-RPE-esclera esquartejado com super fino pinça, retire suavemente as folhas intactas de RPE da membrana basal subjacente (membrana de Bruch) usando uma faca microcirúrgica crescente sob o microscópio de dissecação colocado no fluxo laminar (Figura 1f).<ol>
<li>Transfira as folhas RPE para a nova placa de cultura estéril contendo meio de cultura usando uma micropipeta P200. Quando a transferência de folhas RPE, molhar as pontas de pipeta com meio de cultura pipetando várias vezes para evita a aderência dentro a ponta do tecido. RPE será claramente distinguível de coroide: RPE parece mais amarronzado, enquanto coroide é mais escura e parece pegajoso e fofo.</li>
		<li>Como alternativa, use digestão enzimática e dissociação suave sem fórceps para desanexar o RPE a coroide14.</li>
	</ol>
</li>
	<li>Lavar os lençóis RPE 2 - 3 vezes usando previamente aquecido a 37 ° C médio de crescimento da placa de cultura estéril. Após cada etapa de lavagem, cuidadosamente recolha as folhas RPE, evitando outros tecidos como detritos de retina ou coroide. No final da última lavagem recolher as folhas RPE com uma micropipeta P200 e colocá-los em um novo tubo de microcentrifuga de 1,5 mL. Nota: Não há nenhuma necessidade para Centrifugar as folhas RPE: eles irão sedimentos pela gravidade dentro de 1 min.</li>
	<li>Remova o meio de crescimento extra usando uma micropipeta P200. Ressuspender as células em um meio fresco pré-aquecido e suavemente, Triture-os com uma micropipeta P200. Evite a formação de bolhas. Uma suspensão de célula única é o ideal, mas também evitar demasiada trituração, caso contrário, as células RPE não são capazes de sobreviver. Recomendamos muito suavemente trituração por 40 - 50 vezes.</li>
</ol>5. cultivo de células RPE
<ol><li>As células numa placa de cultura da placa.<ol>
<li>Se as aplicações a jusante das células RPE exigem retenção de sua polaridade, placa o RPE células de 2 ratos em uma inserção de membrana de poliéster 12mm pré-carregado em placas de cultura 12-bem. Chapeamento as células em alta densidade é desejado porque nesse caso, as células RPE são capazes de manter as propriedades originais, tais como o formato hexagonal e pigmentação.</li>
	</ol>
</li>
	<li>Não mova a placa ou mudar o meio de crescimento para as primeiras 72 horas de cultivo. Depois de 3 dias, substitua o antigo médio com o meio fresco de crescimento previamente aquecido. Depois troque o meio de cultura todos os dias. Uma vez que as células RPE confluencia, reduza o FBS meio de crescimento de 2%. Nota: As células podem ser divididas usando tripsina 0,25%. No entanto, após a divisão das células podem perder sua forma hexagonal e pigmentação nas passagens subsequentes. Nós não recomendamos a divisão das células, mas se isso for exigido pelas aplicações a jusante da cultura, tenha em mente que as células primárias não podem ser passadas por tempo indeterminado: eles são conhecidos para diferenciar eliminação após 5-7 passagens.</li>
</ol>

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DS recebeu pesquisa financiamento da Bayer HealthCare e F. Hoffmann-La Roche, Alemanha, Suíça. Os restantes autores declaram sem interesses financeiros concorrentes. Este trabalho é apoiado pela pesquisa para evitar cegueira (subvenções irrestritas para o Wilmer Eye Institute e Universidade de Pittsburgh).  Este trabalho também é apoiado por fundos de start-up para DS de Oftalmologia, Universidade de Pittsburgh.

O protocolo detalhado apresentado permite estabelecimento confiável de rato primário RPE culturas que atingem a confluência após 1 semana e apresentar as principais características RPE como forma hexagonal e pigmentação após 2 semanas. As células RPE obtidas podem ser usadas para um número de aplicações a jusante, tais como vitamina A metabolismo9, fagocitose do fotoreceptor segmentos exterior10 e íon transporte11, células epiteliais po...

O epithelium retinal do pigment (RPE) é uma única camada de células epiteliais polarizadas que se localiza entre a retina neural e a coroide do olho. A funcionalidade das células RPE e a integridade da monocamada RPE são críticos para a visão porque o RPE desempenha um papel importante em vários processos, tais como a manutenção da barreira hemato-retiniana exterior, transporte de água e íons entre a retina e o absorção de coroide, luz, proteção contra o estresse oxidativo, controle do metabolismo de retinoide e fagocitose dos segmentos exteriores dos FOTORRECEPTORES1,2. A localização do RPE na parte de trás do olho, bem como sua função de barreira impedindo medicamentos administrados sistemicamente da passagem do sangue para o humor vítreo, tornam difícil estudar a complexidade do RPE função na vivo. Assim, há uma grande necessidade para o estabelecimento de culturas de células RPE para estudar as células RPE em um ambiente flexível e controlado3,4.
Existem várias linhas de células RPE estabelecidas, fornecendo uma maneira fácil e conveniente de obter e armazenar as células; no entanto, passadas as células têm algumas desvantagens em comparação com as células primárias2,3,4. Primeiro, eles são frequentemente caracterizados por alterações na morfologia celular. Por exemplo, nenhuma das linhas celulares existentes foram encontradas para ser adequado para um estudo confiável das propriedades de barreira de RPE devido a perda do fenótipo de polaridade celular e desaparecimento parcial das junções apertadas4. Além da perda de polaridade e ligações de celular para celular adequadas, as linhas de células RPE rapidamente perdem sua pigmentação devido à ausência das enzimas chave melanogênese no adulto RPE5. A pigmentação pode ser restaurada, mas a análise abrangente do mecanismo de re-pigmentação que inclua uma combinação de microscopia eletrônica de transmissão, gene expressão ensaios análise e químico para confirmar a presença de melanina tem nunca foram realizados6. Uma limitação mais é que as linhas de células RPE tem vida estendida da célula (às vezes - imortalidade) e sob certas condições podem se transformar em células-tronco multipotentes auto renovadora que desanexar do substrato e forma flutuante colônias7, 8. essa limitação torna impossível usar as linhas de células para transplante de experiências3.
Tendo em conta as desvantagens das linhas celulares RPE estabelecidas, culturas de células RPE primárias obtidas de tecidos frescos podem servir como um modelo mais biologicamente relevante para estudar o RPE. Células RPE primárias foram usadas não só para estudar funções específicas do RPE, tais como vitamina A metabolismo9, fagocitose do fotoreceptor segmentos exterior10 e íon transporte11, mas também para estudar Biologia celular básica tais como epiteliais polaridade de célula2 , homeostase dos lisossomos e autofagia12,13.
Nos últimos anos tem havido um número de publicações sobre o estabelecimento de culturas primárias de RPE, indicando um crescente interesse nesta área de pesquisa de14,3,15. Inúmeros protocolos para células humanas RPE e células RPE não-humanos como as células RPE de bovinos e suínas foram publicados16,17,18,19. No entanto, é mais difícil de manipular as células RPE rato devido ao seu tamanho muito menor. Apesar de um grande número de publicações tem descrito protocolos para isolar as células RPE do rato14,20,21, ainda existem muitos pesquisadores lutando para isolar as células RPE sem contaminação de coroide células ou células de detritos de retina neural. Aqui nós apresentamos o protocolo para o estabelecimento de mouse principal cultura de células RPE, incluindo a obtenção dos olhos do rato, dissecção dos olhos e isolamento das folhas de RPE para produzir as células para cultivo. Este protocolo de vídeo seria especialmente útil para pesquisadores que estão começando a trabalhar com culturas primárias de RPE rato e precisa de orientação sobre as técnicas de dissecação.

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

primary cell cultures from the mouse retinal pigment epithelium

O epithelium retinal do pigment (RPE) é um altamente polarizado epitélio multi-funcional que se localiza entre a retina neural e a coroide do olho. É uma única folha de células pigmentadas que são embaladas hexagonalmente e conectadas por junções apertadas. As principais funções do RPE incluem a absorção da luz, fagocitose dos segmentos exteriores galpão fotoreceptor, buffer espacial dos íons, transporte de nutrientes, íons e água, bem como participação activa no ciclo visual. Com tais funções importantes e diversas, é criticamente importante estudar a biologia das células RPE. Um número de linhas de células RPE foram estabelecido; no entanto, passadas e imortalizadas células são conhecidas para perder rapidamente algumas das características morfológicas e fisiológicas das células RPE naturais. Assim, as células primárias são mais adequadas para estudar diferentes aspectos da biologia de pilha RPE e função. Cultura de células RPE primária mouse é muito útil para pesquisadores, desde modelos de mouse são amplamente utilizados em estudos biológicos, no entanto, coletar RPE células de rato também é muito difícil devido ao seu pequeno tamanho. Aqui, apresentamos um protocolo para o estabelecimento de culturas de células RPE mouse principal que inclui Enucleação e dissecção dos olhos e isolamento das folhas de RPE para produzir as células para cultivo. Este método permite a recuperação eficiente da célula. As células RPE obtidas de dois ratos podem chegar a confluência em uma inserção de membrana de poliéster 12mm pré-carregado em placa de cultura, depois de uma semana de cultura e mostrar algumas das propriedades originais de boa-fé RPE células como forma hexagonal e pigmentação após dois semanas de cultura.

A rato RPE cultura primária estabelecida de 2 ratos em 12mm membrana de poliéster inserção atinge 90-95% confluência após 1 semana na cultura. Depois de 2 semanas de cultura, as células alcançou a confluência de 100% e começaram a formar um mosaico de células hexagonais, pigmentadas e bi-nucleated. Por 3 semanas de cultura, as células continuou a formar a forma e pigmentação, no entanto, após 4 semanas uma parte das células tem hiperpigmentadas (Figura...

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

O epithelium retinal do pigment (RPE) é um epitélio multi-funcional do olho. Aqui nós apresentamos um protocolo para estabelecer culturas de célula primária derivadas o RPE murino.

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 ...

primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

Research

JoVE Journal

Biology

20.9K visualizações.  University of Pittsburgh School of Medicine. O epithelium retinal do pigment (RPE) é um epitélio multi-funcional do olho. Aqui nós apresentamos um protocolo para estabelecer culturas de célula primária derivadas o RPE murino.

Vídeo: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

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

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

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

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

Biologia

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

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

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

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

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

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

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

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

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

Primary Cell Cultures from Drosophila Gastrula Embryos

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

Primary Cell Cultures from Drosophila Gastrula Embryos

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

Culturas de células primárias de Drosophila Os embriões Gástrula

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

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, ...

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 ...

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

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

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

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

Ablação Mediada por Nitroreductase/Metronidazole e uma Plataforma MATLAB (RpEGEN) para estudar a regeneração do Pigmento Retinal de Zebrafish Epithelium

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

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

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. ...

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 ...