Este trabalho foi possível graças a Fundação de Pesquisa de células e tecidos humanos, o que torna tecidos humanos disponíveis para a pesquisa. O apoio financeiro para este trabalho foi recebido do Ministério Federal de Educação e Pesquisa (nome concessão: Fígado de Rede Virtual, número de concessão: 0315759) e Hepacult GmbH. Nossos agradecimentos vão também para os assistentes técnicos do Banco de tecidos Grosshadern Hospital para a recolha das amostras do fígado e dos assistentes técnicos do Núcleo instalação de isolamento celular para a realização da perfusão hepática e isolamento dos hepatócitos. Em particular, gostaríamos de agradecer Natalja Löwen para demonstrar este procedimento no vídeo. Finalmente, gostaríamos de agradecer Natalja Löwen e Edeltraud Hanesch para criar as ilustrações para a Figura 1 e as figuras na visão esquemática do vídeo.

1. Preparação da perfusão e isolamento Solutions <ol><li> Preparar as soluções necessárias para a perfusão do pedaço de fígado e o isolamento de hepatócitos de acordo com a Tabela 1. As soluções podem ser armazenadas a 4 ° C até à sua utilização. </li><li> Filtrar em condições estéreis todas as soluções que utilizam um filtro de 0,22 um. </li><li> Todas as soluções que entram em contato com o fígado deve ser estéril. </li></ol> 2. Preparação de Perfusão Equipamentos e Soluções <ol><li> O equipamento para a perfusão do pedaço de fígado deve ser configurado como mostrado na Figura 1. </li><li> O banho de água deve ser ajustado a uma temperatura apropriada, que é diferente em cada um determinado conjunto experimental, de modo a que as soluções são a uma temperatura de 37 ° C quando atingem o pedaço de fígado. Neste caso, o banho de água é ajustado a 41 ° C para aquecer as soluções 1, 2 e 3 e do condensador de vidro encamisado. Solução 4 deve ser aquecido até37 ° C em um banho de água para uso separado para reduzir a perda da actividade da colagenase. </li><li> Pouco antes da perfusão hepática, ligue o regulador do tanque de gás contendo 95% de O 2/5% de CO2 para abastecer o aparelho de oxigenação (Figura 1E). </li></ol> 3. Perfusão do fígado <ol><li> Um pedaço do fígado com tanto intacto cápsula de Glisson quanto possível e de preferência com superfície de apenas 1 corte deve ser obtida a partir de um patologista para perfusão. </li><li> Colocar este pedaço de fígado num funil de Buchner que contém um disco de filtro perfurado (Figura 1B). </li><li> O sistema de perfusão que devem ser iniciadas com a solução 1. </li><li> Com uma baixa taxa de fluxo, as cânulas de irrigação curvo com pontas de azeitona deve ser inserido dentro de vasos sanguíneos maiores na superfície de corte do pedaço de fígado. Como o sangue libera fora do fígado, o tecido se torna mais leve em áreas com boa perfusão. O número de cânulas usadas para vários tamanhosde fígados é mostrado na Figura 2A. O tamanho calibre escolhido deve resultar em um ajuste confortável que irá realizar as cânulas no lugar. Os vasos sanguíneos mais pequenos devem ser deixados em aberto para o tampão de perfusão para drenar para fora do pedaço de fígado. </li><li> Aumentar a taxa de fluxo na bomba peristáltica, a entre 110-460 ml / min, dependendo do tamanho do fígado (Figura 2B). Isto resulta numa velocidade de fluxo média de 44 ± 16 ml / min por uma cânula (Figura 2C). A velocidade escolhida depende do pedaço de fígado e deve resultar num ligeiro plumping do pedaço de fígado. Em alguns casos, pode ser necessário a braçadeira fechada uma parte dos vasos abertos com grampos vasculares micro para atingir o ligeiro plumping mencionado acima. Uma boa perfusão pode ser observada quando o pedaço de fígado é uma cor mais clara em toda. </li><li> Mantenha o pedaço de fígado úmido durante a perfusão, cobrindo-o com um pedaço de gaze embebida em solução salina. </li><li> Perfundir com 1 L de Solução 1 para expulsar qualquer remaining sangue no pedaço de fígado. </li><li> Mude o líquido de perfusão para a Solução 2 e perfuse por 10 min. </li><li> Mude o líquido de perfusão para a Solução 3 e aspergir com 0,5 L. </li><li> Alterar o fluido de perfusão à solução 4, que contém de 0,1-0,15% de colagenase (Tabela 2). </li><li> Para esta etapa, a perfusão deve ser levada a cabo de uma forma de recirculação para 9-12 minutos ou até que o fígado está suficientemente digerida, o tecido de fígado deveriam aparecer a separar ligeiramente sob a cápsula de Glisson e sentir amolecida, quando sondada com o lado sem corte de um bisturi. </li></ol> 4. O isolamento de hepatócitos <ol><li> Desligue a bomba peristáltica e remover cânulas do pedaço de fígado. </li><li> Coloque o pedaço de fígado em um prato de cristalização, contendo 100-200 mL de solução 5. </li><li> Retire a cápsula de Glisson com cuidado e agite para fora das células. Se há regiões que não são bem perfundidos, um bisturi pode ser utilizada para cortar atravésessas regiões para liberar as células contidas. Adicionar mais Solution 5 conforme for necessário durante o processo. </li><li> Adicionar mais 5 solução até um volume final de 500 ml foi atingido. </li><li> Filtrar a suspensão de célula duas vezes: em primeiro lugar através de uma malha de nylon de 210 mM seguido por uma malha de nylon de 70 ^ m. Em seguida, despeje a suspensão de células em tubos de 200 ml de centrífuga. </li><li> Centrifugar a suspensão de células a 72 g durante 5 min a 4 ° C. Aspirar o sobrenadante e ressuspender o sedimento de células suavemente suavemente em 200 ml de solução 5. </li><li> Repita o passo número lavagem 4,6 três vezes. Na etapa final centrífuga, Ressuspender as células em solução de armazenamento a frio (ver lista de materiais). As células devem ser de aproximadamente 2-5.000.000 hepatócitos por mililitro de avaliação de rendimento e viabilidade através de um teste de exclusão de azul de tripan baseada em hemocitômetro. </li><li> Para realizar um ensaio de exclusão com azul de tripano, adicionar 0,1 ml de células diluídas de forma apropriada (≈ 2-5.000.000 / ml) para um tubo de microcentrífuga contendo 0,5 ml de solução que tripano azulem (0,4% de azul de tripano dissolvido em solução salina tamponada com fosfato (PBS)), e 0,4 ml de PBS. Após a mistura da suspensão de células cuidadosamente, carregar um hemocitómetro com a suspensão e examinar ao microscópio a uma ampliação de 100X. Na microscopia, as células mortas será manchada azul, enquanto as células vivas aparecem sem mácula. Contar o número de células vivas e total em cada um dos 1 milímetro 2 grades marcadas no hemocitômetro. Viabilidade (%), rendimento de células vivas (milhões de hepatócitos por ml de solução de armazenamento a frio (CSS) ou milhões de hepatócitos por g de fígado) pode ser calculado através das fórmulas abaixo. </li></ol><img alt="Equação 1" fo:src="/files/ftp_upload/50615/50615eq1highres.jpg" src="/files/ftp_upload/50615/50615eq1.jpg" /> Nota: Neste caso, o factor de diluição do azul de tripano é 10 e o factor de hemocitómetro é 10.000.

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Otimização deste protocolo foi parcialmente financiado por uma concessão do Hepacult GmbH. Dr. Wolfgang Thasler é um dos fundadores da Hepacult GmbH e continua sendo um dos membros do conselho nesta empresa. O emprego de Maresa Demmel é parcialmente por Hepacult. Maria Hauner é empregado por Hepacult GmbH. Hepacult é um spin-off empresa biotecnológica da Universidade, que oferece hepatócitos humanos com o consentimento e acesso aberto para fins de pesquisa.

Este protocolo resulta no isolamento de hepatócitos humanos com alta viabilidade e pureza. Para atingir estes resultados, é importante para começar com um pedaço de fígado adequado. O pedaço de fígado deve ter cápsula intacta de Glisson em todas as superfícies, exceto para 1 superfície de corte. Outro fator importante é o lote especial de colagenase utilizada, como lotes diferentes podem resultar em diferenças marcantes em viabilidades de hepatócitos após digestão 11. Portanto, diferentes lotes...

A correction was made to: <a href="http://www.jove.com/video/50615/isolation-human-hepatocytes-two-step-collagenase-perfusion">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure </a>
The changes listed below have been made to Table 1.
1). In the recipe for Solution 2, the Final Concentration of EGTA has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>0.1mM</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>EGTA&#160;</td>
			<td>1mM</td>
		</tr>
	</tbody>
</table>
2). In the recipes for Solution 3 and 4, the Final Concentration of Calcium chloride dihydrate has been changed from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>0.5&#181;M</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>5mM</td>
		</tr>
	</tbody>
</table>

A correction was made to: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.
The Final Concentration of EGTA in Solution 2 and the Final Concentration of Calcium chloride dihydrate in solutions 3 and 4 have been updated in Table 1.

Erratum: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

A suspensão de células de fígado pode ser preparado a partir do fígado por meio de métodos mecânicos ou enzimáticos. Os métodos mecânicos usados ​​para preparar as células do fígado integrais incluem forçando o fígado através de gaze 2, agitando um pedaço de fígado com contas de vidro em uma coqueteleira Kahn 3, utilizando homogeneizadores de vidro com pilões soltas 4,5 etc Ao longo dos anos, os métodos mecânicos caíram fora do favorecem, devido à lesão das membranas das células e a perda da função dos hepatócitos isolados 6,7. Por conseguinte, a utilização de um método enzimático é actualmente o principal método para o isolamento de hepatócitos. Isolamento de hepatócitos, utilizando um método enzimático aumentou quando Berry e amigo 8 perfusão colagenase e hialuronidase através do fígado através da veia porta em ratos. Este processo de perfusão utilizado vasculatura para permitir que os enzimas a entrar em contacto estreito com a maioria das células, o que leva aum aumento de 6 vezes no rendimento de hepatócitos 8. Além disso, este método produziu células que retinham a sua integridade estrutural, com virtualmente nenhuma transformação do retículo endoplasmático em vesículas isoladas e sem dano mitocondrial 8. Este método foi modificado por Seglen 1, que foi pioneiro de um procedimento de perfusão de duas etapas para o isolamento das células do fígado. Neste procedimento, o fígado de rato é perfundido com uma Ca2 + livre tampão seguido por perfusão com um tampão de colagenase contendo Ca 2 + 1. A remoção de Ca 2 + no primeiro passo ajuda a romper desmossomas, enquanto a adição de Ca 2 + no segundo passo é necessário para a actividade da colagenase ideal 1,9. Dado que os trabalhos publicados acima descrito foi realizado em ratos, este artigo destina-se a demonstrar um procedimento modificado, que pode ser usado para isolamento de hepatócitos com alta viabilidade de zumbidoum fígado. O uso de hepatócitos humanos continua a ser importante para a investigação de translação e para a validação de experimentos com modelos animais. Os pedaços de fígado humano utilizados neste estudo foram adquiridos com o consentimento para a governança por meio da Fundação de Pesquisa de Tecidos Humanos e celular, uma fundação sem fins lucrativos controlada pelo Estado 10. Depois de um patologista removido o que era necessário para o diagnóstico, pedaços de fígado foram coletadas a partir do tecido remanescente. O tecido seccionado pelo patologista foi morfologicamente tecido saudável obtida a partir de margens de ressecção após a ressecção hepática.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass jacketed condenser</td>
			<td>Ga&szlig;ner Glastechnik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>41 &deg;C Water bath</td>
			<td>Julabo</td>
			<td>35723-H24/EG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37 &deg;C Water bath</td>
			<td>GFL</td>
			<td>1083</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compressed gas cylinder (95 % O2/5 % CO2)</td>
			<td>Linde</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas permeable tubing</td>
			<td>Neolab</td>
			<td>2-4440</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td>IP65</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>320010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Omnilab</td>
			<td>5171014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 1 L</td>
			<td>Schott Duran</td>
			<td>2121654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Conical flasks 5 L</td>
			<td>Schott Duran</td>
			<td>2121673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beakers</td>
			<td>Schott Duran</td>
			<td>2110654</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 ml centrifuge tubes</td>
			<td>Becton Dickinson</td>
			<td>352075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Omnilab</td>
			<td>5144063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Curved irrigation cannulae with ball tips</td>
			<td>Ernst Kratz GmbH</td>
			<td>1464LL/ 1465LL A+B/ 1472LL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro vascular clamps</td>
			<td>Ernst Kratz GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>Carl Roth</td>
			<td>HT38.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 210 &mu;m</td>
			<td>Neolab</td>
			<td>4-1413</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nylon mesh 70 &mu;m</td>
			<td>Neolab</td>
			<td>4-1419</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.22 &mu;m sterile filters</td>
			<td>Peske</td>
			<td>99505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>500 ml bottles</td>
			<td>Schott Duran</td>
			<td>2180144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 L bottles</td>
			<td>Schott Duran</td>
			<td>2180154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Peske</td>
			<td>06-0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>0030 120,086</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Neolab</td>
			<td>1508454</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile Pasteur pipettes</td>
			<td>Brand</td>
			<td>747715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Motorised pipette filler (Pipette boy acu)</td>
			<td>Integra</td>
			<td>155017</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Refridgerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar flow</td>
			<td>Kendro</td>
			<td>Hera safe-KS9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspirator (Low-flow surgical suction pump)</td>
			<td>Atmos</td>
			<td>C361</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory Gas Burner</td>
			<td>Integra</td>
			<td>Fire Boy eco</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable laboratory coat</td>
			<td>Paperlynen GmbH</td>
			<td>MD0202414</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical mask with visor</td>
			<td>Kimberly-Clark</td>
			<td>48247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical hood</td>
			<td>Barrier</td>
			<td>42072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Semper Care</td>
			<td>CE0321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase (Batch number NB 4G)</td>
			<td>Serva</td>
			<td>17465</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck</td>
			<td>2382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>9265.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Roth</td>
			<td>9105.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Serva</td>
			<td>26868</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Biomol</td>
			<td>01400-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Serva</td>
			<td>22700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Serva</td>
			<td>30180</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.4% Trypan blue solution</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cold storage solution</td>
			<td>Hepacult GmbH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation of human hepatocytes by a two-step collagenase perfusion procedure

O fígado, um órgão com capacidade de regeneração excepcional, realiza uma ampla gama de funções, como a desintoxicação, metabolismo e homeostase. Como tal, os hepatócitos são um modelo importante para uma grande variedade de questões de pesquisa. Em particular, o uso de hepatócitos humanos é especialmente importante nas áreas de farmacocinética, a toxicologia, a regeneração do fígado e investigação de translação. Assim, este método apresenta uma versão modificada de um procedimento de perfusão com colagenase em dois passos para isolar hepatócitos como descrito por Seglen 1. Anteriormente, os hepatócitos foram isolados por métodos mecânicos. No entanto, os métodos enzimáticos têm sido mostrados como sendo superior como hepatócitos mantêm a sua integridade estrutural e a função, depois do isolamento. Este método aqui apresentado adapta o método concebido anteriormente para os fígados de ratos aos pedaços de fígado humano e resulta num grande rendimento de hepatócitos com uma viabilidade de 77 ± 10%. A principaldiferença neste processo é o processo de canulação dos vasos sanguíneos. Além disso, o método aqui descrito também pode ser aplicado a partir de fígados de outras espécies com tamanhos do fígado ou dos vasos sanguíneos comparáveis.

Configuração de Perfusão Os equipamentos necessários para a perfusão hepática deve ser configurado de acordo com a Figura 1. Viabilidade e Rendimento de isolado hepatócitos humanos A viabilidade média de hepatócitos humanos isolados foi de 77 ± 10% eo rendimento médio dos hepatócitos foi de 13 ± 11 milhões de hepatócitos / g de fígado, com valores expressos em média ± desvio padrão. O número de isolament...

Watch this Scientific Journal Video about Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure at JoVE.com

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Um procedimento de duas etapas de perfusão de colagenase modificado para isolamento de hepatócitos humanos é descrita. Este método também pode ser aplicado a outros fígados de mamíferos. Os hepatócitos isolados estão disponíveis em alto rendimento e viabilidade, tornando-os um modelo adequado para a investigação científica em áreas como a regeneração do fígado, farmacocinética e toxicologia.

Isolamento de hepatócitos humanos por duas etapas Collagenase Perfusão Procedimento

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

isolation-human-hepatocytes-two-step-collagenase-perfusion

Research

JoVE Journal

Biology

30.8K visualizações.  Grosshadern Hospital, Munich.  Um procedimento de duas etapas de perfusão de colagenase modificado para isolamento de hepatócitos humanos é descrita. Este método também pode ser aplicado a outros fígados de mamíferos. Os hepatócitos isolados estão disponíveis em alto rendimento e viabilidade, tornando-os um modelo adequado para a investigação científica em áreas como a regeneração do fígado, farmacocinética e toxicologia. 

Vídeo: Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Biologia

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento de fibroblastos de rato por Portal<em&gt; In situ</em&gt; Perfusão de fígado

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease Neutra

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-Sua purificação: A Two-step Affinity Purification Protocolo Cedendo Full-length proteínas purificadas

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Exame<em&gt; Drosophila</em&gt; Larval células terminais traqueais por microscopia de luz

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocolo para o isolamento de hepatócitos humanos primários e Correspondente principais populações de células do fígado não-parenquimatosas

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Análise de microbiota usando PCR de duas etapas e sequenciamento do gene 16S rRNA de próxima geração

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Uma estratégia de duas etapas que combina modificação epigenética e sinais biomecânicos para gerar células pluripotentes mamíferas

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Isolamento de Hepatócitos Primários de Ratos com Controle de Perfusão Multiparâmetro

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Quantificação aprimorada de proteínas usando isolamento mitocondrial em duas etapas

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Autor em destaque: Isolamento sem marcação em duas etapas de mitocôndrias para melhor descoberta e quantificação de proteínas  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.