The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

Os animais foram mantidos no Centro de Medicina Experimental (FEM, Charité, de Berlim, Alemanha), e todos os protocolos experimentais foram revistos e aprovados pela Secretaria de Estado da Saúde e Assuntos Locais (LAGeSo, Berlim, Alemanha; Reg. No. 0365 O / 11). Colheita 1. Fígado <ol><li> Preparação pré-cirúrgica <ol><li> Use uma placa de cortiça para fixação. Coloque uma cortina médico sobre a placa. Usando quatro agulhas, fixar uma máscara de inalação na placa de cortiça para narcose intraoperatória inalação. </li></ol></li><li> Prearrangement do Portal venosa cânula <ol><li> Conecte-se um cateter venoso periférico (G 20) a uma torneira de três vias. O cateter venoso deve ser cortado obliquamente, com um comprimento de aprox. 1,5 cm. </li><li> De-ar do sistema usando uma seringa de 10 ml cheia com solução de Ringer. É muito importante para cuidadosamente-air de embolia gasosa, porque pode afetar negativamente o decellularization perfusão. </li></ol></li><li> Prearrangementda cânula arterial <ol><li> Usar uma cânula borboleta e cortar a agulha com um comprimento de 5 mm. Instale um tubo de 5 centímetros (ID 0,58 milímetros; OD 0,96 milímetros) para a agulha 5 mm e corrigi-lo com supercola. Inserir um tubo 2 cm de comprimento (ID 0,28 milímetros; OD 0,61 milímetros) para dentro do tubo maior e corrigi-lo com supercola. </li><li> Deslizamento outro tubo sobre essa construção como um pilar e corrigi-lo com supercola. De-ar da cânula arterial com uma seringa de 5 ml cheio com solução de Ringer. </li></ol></li><li> Anestesia e Analgesia <ol><li> Induzir narcose do rato na câmara de indução da anestesia de 3,5% de isoflurano em oxigénio a um caudal de 1,5 L / min. </li><li> Para assegurar a analgesia segura durante a operação, metamizol injectar (100 mg / kg de massa corporal) e a cetamina em dose baixa / medetomidina (10 mg / 0,1 mg por kg de massa corporal) por via intraperitoneal. </li><li> Fornecer 1,5% de isoflurano em 1 L / min de oxigênio via máscara de inalação para posterior manutenção da anestesia cirúrgica. </li></ol></li><li> Arranjos pré-operatórios &lt;ol&gt; <li> Raspar o abdômen liberalmente com uma máquina de corte elétrica. Humedecer o local da incisão no abdómen rapado com etanol a 70% para desinfecção. </li><li> Colocar o animal em decúbito dorsal sobre a placa preparado, insira a cabeça para dentro da máscara de inalação e corrigir os membros com fita adesiva médica e pinos. Retire o excesso de etanol e de pele com uma compressa de gaze. </li></ol></li><li> Avaliação anestesia <ol><li> Avaliar a profundidade da anestesia, observando a freqüência respiratória. Quando a anestesia parece suficientemente profundo (40-60 respirações / min), verifique se a analgesia é suficiente, tentando provocar o reflexo toe pitada usando uma pinça cirúrgica para comprimir a pele entre os dedos de ambas as patas traseiras. </li></ol></li><li> Abordagem abdominal <ol><li> Adicione uma incisão mediana na parede abdominal caudal acima da bexiga. Cortar a partir deste ponto para ambos os arcos hipocôndrios de uma maneira em forma de V. Tome cuidado para não cortar o diafragma. Puxe a parede abdominal corte sobre o peito. </li&gt; <li> Fixar o xifóide com uma braçadeira Overholt, puxe-cranial e corrigi-lo com um elástico. Use cotonetes molhados para evacuar o intestino. Enrole intestino em uma atadura de gaze molhada. Além disso, cobrir todas as margens abdominais com compressas húmidas. </li></ol></li><li> Preparação fígado <ol><li> Dissecar o ligamento falciforme. Use uma atadura de gaze molhada para manter os lobos hepáticos medial e cranial esquerda sob a cúpula do diafragma. Expor o ligamento hepatoduodenal eo lóbulo do fígado omental superior. </li><li> Use duas micropinças e tesoura primavera para dissecar cuidadosamente o ligamento anexado ao lobo omental superior até que o lobo é móvel. Mova o lobo usando cotonetes molhados sob a compressa de gaze, segurando os lobos medial e deixados no local. </li><li> Depois de mobilização do lóbulo do fígado omental superior, passar do estômago para a esquerda. Fixar o estômago com uma braçadeira e dissecar o omento menor com micropinças e os tesoura primavera. </li><li> Mobilizar a omentos menorl lóbulo do fígado até que ele é móvel e, posteriormente, mover o lobo de volta em sua cavidade. Usar uma braçadeira Backhaus para reter o duodeno. Mover o duodeno para a esquerda para esticar e expor o ligamento hepatoduodenal. Circunscrever o ducto biliar comum no ligamento hepatoduodenal esticado. </li><li> Dissecar o ducto biliar do tecido adiposo e no pâncreas. Corte o ducto biliar 1,5 cm do bifurcação do ducto biliar. Dissecar a veia porta a partir do tecido adiposo circundante. Expor a veia gastroduodenal. </li><li> Ligadura da veia gastroduodenal duas vezes com seda 6-0 e dividir a veia entre as duas ligaduras. Dissecar o tronco celíaco e da artéria hepática comum do tecido circundante até que todos rami arterial são revelados. </li><li> Libertar a artéria gastroduodenal a partir do tecido circundante. Ate a artéria gastroduodenal duas vezes com seda 6-0, com os laços distantes um do outro. Dissecção da artéria gastroduodenal entre as duas ligaduras. </li><li> Libere a artéria esplênicaa partir do tecido circundante. Amarre a artéria esplênica duas vezes com seda 6-0, com os laços distantes uns dos outros. Dissecção da artéria esplênica entre as duas ligaduras. </li><li> Libertar a artéria gástrica esquerda a partir do tecido circundante. Amarre a artéria gástrica esquerda duas vezes com seda 6-0, com os laços distantes uns dos outros. Dissecção da artéria entre as duas ligaduras. </li><li> Circunscrever a veia cava inferior entre o rim direito e no lobo hepático lateral direito. Dissecar a veia do espaço retroperitoneal. Expor a aorta, seguindo o tronco celíaco. </li><li> Dissecar o tecido adiposo retroperitoneal. Injectar 1000 IE de heparina em 1 ml de solução salina na veia cava inferior. Retrair a cânula e fechar a incisão com um algodão. Espera 1-2 min para o efeito sistémico de heparina. </li><li> Circunscrever a aorta com uma pinça. Dissecar a aorta e desangrar o rato. Além disso, dissecar a veia cava inferior distalmente a partir do rim. </li></ol></li><li> Portal venoso Canulação (Use a cânula preparada na secção 1.2) <ol><li> Faça uma incisão na boca peixe na veia portal distal. Abra a incisão com uma pinça e canular a veia porta. Lave o fígado com 20 ml de solução de Ringer até os descolore fígado e fixar a cânula com ligaduras de seda (6-0). </li></ol></li><li> A canulação arterial (Use a cânula preparada no ponto 1.3) <ol><li> Dissecar a aorta acima do tronco celíaco. Libere o segmento de aorta a partir do espaço retroperitoneal. Corte o segmento de aorta longitudinalmente para gerar um remendo aórtico. Insira a cânula auto-construído em um suporte estático. </li><li> Use uma pinça para deslizar 2 a correção da aorta através da cânula. Ligadura da artéria hepática com seda 6-0 sobre a cânula e corrigir o patch no pilar. </li></ol></li><li> Explantation fígado <ol><li> Abra o diafragma, fazer uma incisão circular e dissecar o fígado a partir das estruturas circundantes. </li></ol></li><li> Armazenamento <ol><li> Store o fígado num tanque estéril de 500 ml cheio com 350 ml de solução de Ringer até à sua utilização. </li></ol></li></ol> 2. As soluções de detergentes <ol><li> Solução Triton X-100 estoque 10% <ol><li> Encha uma garrafa de 1000 ml com 900 ml água destilada. e adiciona-se 100 ml de Triton X-100 (99,9%). Usar um agitador para dissolver o Triton X-100. Encha a garrafa com água destilada. até um volume total de 1000 ml foi atingido. </li></ol></li><li> 1% de Triton X-100 <ol><li> Adiciona-se 100 ml da solução de reserva (10%) a um frasco de 1000 ml. Adicionar 900 ml de água destilada. e agitar o frasco duas vezes. Filtra-se a solução a 1% de Triton X-100 através de um filtro estéril. </li></ol></li><li> SDS a 10% da Solução <ol><li> Encha uma garrafa de 1000 ml com 900 ml água destilada. e adicionar 66,99 g de SDS (99,9%, densidade 0,67). Usar um agitador para dissolver o SDS. Encha a garrafa com água destilada. até um volume total de 1000 ml foi atingido. </li></ol></li><li> SDS a 1% <ol><li> Adiciona-se 100 ml da solução de reserva (10%) de um bot de 1000 mlTLE. Adicionar 900 ml de água destilada. e agitar o frasco duas vezes. Filtra-se a solução de SDS a 1%, através de um filtro estéril. </li></ol></li></ol> 3. Decelularização <ol><li> Set-up de Perfusão Dispositivo <img alt="Figura 1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> Figura 1:. Esquema do aparelho de perfusão para a Seletiva Arterial e Venoso Portal Perfusão de quatro fígados de rato sob oscilantes Condições Pressão (. Modificado a partir Struecker et al 14) <ol><li> Vincular o dreno para uma torneira de três vias e uma extensão Heidelberger para regular o nível de preenchimento e preencha o reactor com 500 ml de NaCl 0,9%. <img alt="Figura 2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> Figura 2:. Esquema da perfusão set-up Ligar a câmara de perfusão (1,400 centímetros 3) (1) à extremidade distai (4) do borbulhador (5) através do acesso venoso portal (2) ou por meio da ac arterialcesso (3). O borbulhador (5) deve ser equipado com uma obturação (4), na extremidade distai e uma abertura (6). Ligue o borbulhador (5) para uma extensão de Heidelberger (7). Ligar a extensão Heidelberger para o segmento de bomba (8). Além disso, ligar o segmento de bomba (8) para o outro ramal de Heidelberger (9). Inserir a última extensão Heidelberger (9) para dentro do frasco de detergente (10). Ligue o respirador (11) para a câmara de perfusão (ou para o distribuidor de pressão no caso de múltiplas perfusões). Para drenar os fluidos do reator, conecte a saída para o frasco de resíduos (12). <ol><li> Inserir o segmento de bomba para dentro da bomba. Iniciar a bomba para encher o ciclo com PBS, fechar a extremidade distal do borbulhador e abrir o respiradouro. Quando o fundo da bolha está firmemente coberto com PBS (03/02 centímetros), a fechar a abertura e abrir a extremidade distal do borbulhador. </li><li> Instalação de fígado <ol><li> Ligar a torneira de três vias da cânula da veia porta para o reactor de acesso relacionados. CONECTE da cânula arterial para o acesso arterial. Tenha cuidado para garantir que nenhum ar entra nos sistemas. </li></ol></li><li> Aplicação de oscilantes Condições Pressão <ol><li> Use uma máscara com uma frequência de 15 bpm e uma amplitude de 26 mbar (min. 4 mbar, 30 max. Mbar). Ligue o respirador para a câmara de distribuição de pressão e ligar a câmara ao reactor. Iniciar a aplicação de pressão. </li></ol></li><li> Decelularização Protocolo <ol><li> Execute as etapas de perfusão com um caudal de 5 ml / min. Iniciar a descelularização com a perfusão do fígado com 1% de Triton X-100 durante 90 min. Permitir effluence resíduos através do fluxo de saída do dispositivo de perfusão para manter o nível do fluido no interior da câmara de perfusão e constante sobre o fígado perfundido. Após 90 min, alterar o detergente a 1% de SDS. <img alt="Figura 3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="Tabela 1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;p&gt; Figura 3: Protocolo de Perfusão para todos os grupos experimentais.

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The authors have nothing to disclose.

Embora a técnica apresentada para a colheita de fígado de rato e descelularização é facilmente reprodutível, existem certos passos críticos a serem considerados: Durante a preparação para a colheita do fígado, é importante para evitar o sangramento grave, porque ele vai activar a coagulação do sangue e podem conduzir à formação de coágulos sanguíneos dentro do fígado. Em nossa opinião, é vantajoso entalhar a aorta abdominal directamente antes da canulação da veia porta...

Descelularização e recelularização pode permitir a geração de órgãos funcionais, transplantáveis ​​in vitro 1. Ao remover as células e material antigénico (por exemplo., ADN, epítopos alfa-Gal) de um órgão, a matriz extracelular ou menos não-imunogénico (ECM) pode ser obtido. Esta matriz conserva a microanatomy tridimensional de um órgão e podem servir como a biomatriz ideal para o repovoamento com células de uma origem diferente, possivelmente xenogénico 2. Assim, uma matriz de fígado de rato descelularizado poderia ser repovoada com células de fígado humano. Este micro-fígado humanizado poderia servir como um modelo ex vivo para a pesquisa sobre doenças (por exemplo., Doenças metabólicas, doenças virais inatos ou neoplasias) ou para testes pré-clínicos farmacêutica 3. Vários protocolos diferentes para perfusão de fígado de rato decellularization já foram publicados 4-13. Em todos os protocolos, decellularção foi conseguida por perfusão de detergentes iónicos ou não-iónicos alcalinas através da veia portal canulada. Para o melhor de nosso conhecimento, fomos o primeiro grupo a relatar rato decellularization fígado por perfusão seletiva através da veia portal e / ou o rato artéria hepática 14. Activar a perfusão selectiva dos diferentes sistemas vasculares no fígado pode permitir melhores resultados decelularização e, além disso, podem desempenhar um papel importante no repovoamento celular. No estudo detalhado aqui, fígados foram perfundidos em um dispositivo de perfusão proprietária feitos sob medida, permitindo a perfusão sob condições de pressão oscilantes. Estas condições de pressão imitar a perfusão fisiológica respiratória dependente do fígado: in situ, o fígado paira sob a cópula do diafragma, cujo movimento durante a respiração tem um impacto direto sobre a perfusão hepática. Inspiração conduz especificamente a redução do diafragma e apertando do fígado, optimizandosaída hepato-venoso, enquanto que leva a expiração do fígado elevação e abaixamento da pressão intra-abdominal para optimizar o fluxo venoso portal-15. O nosso objectivo foi avaliar se a condições de pressão oscilante tem um impacto sobre a homogeneidade de fígado de rato descelularização perfusão intra-abdominais, imitando as condições ex vivo. A homogeneidade do processo de descelularização pode ser um fator subestimado na perfusão descelularização. Todos os agentes conhecidos utilizado para a descelularização fígado provocam alterações nas ECM. Células em áreas mal perfundidos permanecer dentro da ECM, ao passo que outras áreas já estão completamente descelularizados. Para se dissolver as células restantes, a duração ou a pressão de perfusão deve ser elevada, fazendo com que mais alterações às áreas bem perfundidos. Assim, detergentes para a descelularização deve ser distribuído de forma homogénea dentro do órgão.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Self built arterial cannula</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/100</td>
			<td>0,28mm ID 0,61mm OD</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/200</td>
			<td>0,58mm ID 0,96m OD</td>
		</tr>
		<tr>
			<td>Venodrop Safe butterfly catheter</td>
			<td>Fresenius Kabi</td>
			<td>3275851</td>
			<td>21 G</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>portal vein cannula</td>
		</tr>
		<tr>
			<td>Periphereal Venous Catheter</td>
			<td>BD</td>
			<td>393224</td>
			<td>BD Venflon Pro 20G</td>
		</tr>
		<tr>
			<td>three-way stopcock</td>
			<td>smiths medical</td>
			<td>888-101RE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Cotton Sticks</td>
			<td>Hecht-Assistent</td>
			<td>4302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pads&#160;</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>13H118-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Bandage</td>
			<td>Hubei Haige&#160; Medical Instruments</td>
			<td>14388</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ringer Solution</td>
			<td>Fresenius Kabi</td>
			<td>13 HKP022</td>
			<td>1000ml</td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10ml/ Luer Solo</td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606061V</td>
			<td>5ml /Luer Solo</td>
		</tr>
		<tr>
			<td>Suture (Silk 6/0)</td>
			<td>Resorba</td>
			<td>H1F</td>
			<td>LOT 105001.81</td>
		</tr>
		<tr>
			<td>medical drape</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>D0613011</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Geuder</td>
			<td>17570</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>Inox-Electronic</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-scissors</td>
			<td>Martin</td>
			<td>11-740-11</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>S&#38;T&#160;</td>
			<td>112314</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Aesculap</td>
			<td>BH111R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>F S T&#160;</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Aesculap</td>
			<td>BD 557</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Decellularisation</td>
		</tr>
		<tr>
			<td>Respirator</td>
			<td>Resmed</td>
			<td>14.24.11.0004</td>
			<td>SmartAIR ST</td>
		</tr>
		<tr>
			<td>Perfusion Device</td>
			<td>Charite, medical engineering laboratory</td>
			<td>custome-made device</td>
			<td>decellularisation device</td>
		</tr>
		<tr>
			<td>peristaltic pump ismatec reglo ICC</td>
			<td>IDEX</td>
			<td>ISM4408&#160;</td>
			<td>4-channel</td>
		</tr>
		<tr>
			<td>heidelberger extension 75 cm&#160;</td>
			<td>Fresenius Kabi</td>
			<td>2873</td>
			<td>75 cm</td>
		</tr>
		<tr>
			<td>MS/CA pump-segment</td>
			<td>IDEX</td>
			<td>IS 3510&#160;</td>
			<td>MS/CA/click&#39;n&#39;go/POM-C</td>
		</tr>
		<tr>
			<td>CA 2-stopper tube</td>
			<td>Pharmed</td>
			<td>BPT NSF-51</td>
			<td></td>
		</tr>
		<tr>
			<td>bubble trap&#160;</td>
			<td></td>
			<td>custome-made item</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock hose connector</td>
			<td>Neolab</td>
			<td>No. 02-1887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detergents</td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth&#160;</td>
			<td>CN30.4&#160;</td>
			<td>2,5 kg</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth&#160;</td>
			<td>3051.1&#160;</td>
			<td>10l</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>staining</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer hematoxylin</td>
			<td>AppliChem</td>
			<td>A4840</td>
			<td></td>
		</tr>
		<tr>
			<td>gomori staining</td>
			<td>Morphisto</td>
			<td>11104</td>
			<td></td>
		</tr>
		<tr>
			<td>AlcainBlue-PAS staining</td>
			<td>Morphisto</td>
			<td>11388</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Red 80&#160;</td>
			<td>Sigma Aldrich</td>
			<td>365548</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for decellularization of rat livers in an oscillating-pressure perfusion device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

A homogeneidade e, assim, a eficácia de diferentes protocolos de decelularização foram avaliadas por observação macroscópica, análise histológica e análise do conteúdo de ADN permanecendo dentro do fígado descelularizados matrizes. Além disso, a carcaça corrosão foi realizada para visualizar a microanatomia intacta de fígados após a descelularização. Macroscopia Durante decellularization, fígados tornar Lucent, indicando a remoção de conteúd...

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Procedimento para Decelularização de fígados de rato em uma Perfusão dispositivo oscilante pressão

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Research

JoVE Journal

Bioengineering

10.2K visualizações.  Charité – Universitätsmedizin Berlin. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Vídeo: Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decelularização e Recellularization de Fígados inteiros

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Bioengenharia

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedimento para decelularização do Coração Suínos por perfusão coronariana retrógrada

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Um instrumento ultra-sônico para condução nervosa bloquear em modelos de ratos diabéticos

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Desenvolvimento de um Biosensor eletroquímico do DNA para detectar um patógeno de origem alimentar

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Uma nova técnica cirúrgica como fundamento na Vivo parcial engenharia fígado de rato

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Decellularization do coração humana dentro de uma bolsa pressurizada em uma orientação invertida

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Geração de Condições Ambientais Dinâmicas usando um dispositivo microfluido de alto rendimento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Aquisição e Perfusão-Descelularização de Flaps Vascularizados Suínos em Biorreator de Perfusão Customizado

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Captação e Descelularização de Membros Posteriores de Rato Usando Biorreator Ex Vivo à Base de Perfusão para Alotransplante Composto Vascularizado

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...