This work was supported by The British Heart Foundation and The Oak Foundation.

Executar todas as experiências com animais de acordo com protocolos aprovados pelo Comitê Institucional de uso e cuidados de animais de laboratório. 1. Preparação de Meios de Cultura <ol><li> Adicione meios de cultura para a linha de células de fibroblasto humano CCL-153: F-12K Médio, 10% de soro fetal bovino (FBS) e 100 U / ml de penicilina e estreptomicina. </li><li> Adicione reprogramação dos meios para geração de PPCPE: meio de Eagle modificado por KO Dulbecco (DMEM) contendo 20% de substituição KO Soro, 0,1 mM β-mercaptoetanol, 0,1 Mínimo mM Meio Essencial (MEM) não-aminoácidos essenciais, 10 ng / ml do factor de crescimento básico de fibroblastos 2 (bFGF-2) e 100 U / ml de penicilina e estreptomicina. Adicionar bFGF-2 recém cada vez antes de usar a mídia. </li><li> Adicione Diferenciação Meios (DM) para induzir células de músculo liso (SMC) diferenciação: α-MEM meio contendo 10% de FBS, factor de crescimento de 0,1 mM β-mercaptoetanol, 100 U / ml de penicilina e de estreptomicina e 25 ng / ml Derivado de Plaquetas(PDGF-ββ). </li><li> Adicione endotelial Diferenciação Meios (CE-MS) para induzir a diferenciação de células endoteliais (CE): crescimento endotelial meios-2 (EGM-2) meio contendo 50 ng / mL de Factor de Crescimento Endotelial Vascular (VEGF) e 100 U / ml de penicilina e estreptomicina. </li></ol> 2. reprogramação fibroblastos humanos em induzidas Parcialmente células-tronco pluripotentes (PPCPE) <ol><li> Cultura linha de células de fibroblastos humanos CCL-153 em 0,04% de solução de gelatina frascos revestidos em meio de cultura. </li><li> Células de passagem a cada 3 dias, à razão de 1: 6. Use as células antes da passagem 9 para otimizar a eficiência da geração de PPCPE. As células estão prontas para transfecção ao atingir 80 - 90% de confluência. </li><li> Linearizar pCAG2LMKOSimO plasmídeo polycistronic contendo 4 fatores de reprogramação octâmero vinculativo fator de transcrição 4 (OCT4), SOX2, fator Kruppel-like 4 (KLF4) e C-MYC (pCAG-OSKM). Digest 5 ug de plasmídeo com 5 unidades de enzima de restrição Pvul durante 3 horas a 37 ° C. Purificaro plasmídeo com um kit comercial de acordo com o protocolo do fabricante. </li><li> Transfectar 2 x 10 6 de fibroblastos humanos com 4 ug de plasmídeo linearizado pCAG-OSKM por electroporação com o kit de fibroblasto dérmico humano (NHDF) nucleofection de acordo com o protocolo do fabricante. Suavemente semear as células transfectadas para pré 0,04% de gelatina revestida frasco T25 contendo 5 ml de pré-aquecido reprogramação mídia. </li><li> Após 24 h, alterar forma com suplementos de 25 ug / ml de neomicina para seleccionar uma população pura de células transfectadas. </li><li> Alterar médio em dias alternados até dia 4. Nota: fibroblastos humanos tornam-se PPCPE após 4 dias de reprogramação. </li></ol> 3. Diferenciação de PPCPE em endotelial e nas células musculares lisas <ol><li> PPCPE Semente em pratos colágeno IV pré-revestidos contendo DM por 4 dias para induzir a diferenciação em células musculares lisas. Alterar médio em dias alternados até dia 4. </li><li> PPCPE Semente sobre o colágeno IV pré-revestidas pratos containing endotelial-DM durante 6 dias para induzir a diferenciação em células endoteliais. Alterar médio em dias alternados até dia 6. </li></ol> 4. descelularizado Aorta Graft Preparação Nota: Todas as soluções e equipamento deve ser estéril. <ol><li> Sacrifique o mouse por deslocamento cervical e corrigir o mouse em uma posição supina sob dissecção microscópio. </li><li> Corte através do esterno e em torno da caixa torácica para abrir a cavidade torácica. Retirar o coração, pulmões e esôfago para expor a aorta. Com cuidado, retire a gordura peri-aorta com uma pinça. </li><li> Corte a aorta a partir do final anterior com uma tesoura e gentilmente segure a ponta com uma pinça sem corte. Retire a aorta da coluna vertebral para trás por dissecção romba. Feche cuidadosamente todas as artérias se ramificam a partir da aorta por ligação com um electrocoagulador bipolar e corte a partir da extremidade da ligação com uma tesoura. </li><li> Use uma seringa de 5 ml para irrigar o lúmen da aorta com 3 ml de solução salina contendo 100 U de heparina para prevenir a formação de coágulos sanguíneos. </li><li> Corte a extremidade posterior da aorta antes de se ramifica na renal. Manter a integridade da aorta é muito importante durante todo o procedimento. </li><li> Lave o lúmen da aorta ascendente com 5 ml de solução de 0,075% de dodecil sulfato de sódio (SDS), diluída em tampão fosfato salino (PBS). </li><li> Mergulhe a aorta torácica em solução SDS 0,075% em uma placa de Petri de 10 cm. Colocar a placa de Petri num agitador orbital durante 2 horas a 150 rpm à temperatura ambiente. </li><li> Lave o lúmen da aorta com 5 ml de PBS. </li><li> Mergulhe a aorta na PBS em uma placa de Petri de 10 cm. Colocar a placa de Petri num agitador orbital durante 2 horas a 150 rpm à temperatura ambiente. Refresque PBS a cada 20 min. </li><li> Lave o lúmen da aorta com 5 ml de PBS. </li><li> Manter enxerto aorta descelularizado em PBS a 4 ° C durante até uma semana. </li></ol> 5. Dupla Sementeira de PPCPE e Bioreactor Condicionado Nota: Toda a soluçãos e o equipamento deve ser estéril. Realizar todas as operações em uma capa de cultura de tecidos. <ol><li> Montar o circuito de fluxo biorreactor, como ilustrado na Figura 1. Ligue câmara de incubação, meios reservatório, bomba peristáltica, e câmara de conformidade com tubagem. Volume mínimo de meio necessário para a circulação é de 40 ml. </li><li> Pré-condição do navio descelularizado com meio de cultura por imersão da aorta em meio DM em um prato de Petri de 10 cm. Colocar a placa de Petri num agitador orbital durante 1 h, a 50 rpm à temperatura ambiente. </li><li> Inserir um centímetro de comprimento de nylon tubos (OD 0,9 milímetros, 0,75 milímetros ID) em ambas as extremidades do recipiente de descelularização sob microscópio. Amarre a embarcação e os tubos com 8-0 suturas de seda. </li><li> Montar o enxerto descelularizado aorta na câmara de incubação, ligando os tubos de nylon com as portas de entrada e de saída (1/32 &quot;) de tubagem a partir da parede da câmara. Manter a câmara de incubação com 5 ml de meio de cada vez. </li><li> Trypsinize PPCPE adicionando pré-aquecidotripsina para cobrir a placa de cultura e agite levemente o prato 10 vezes. Descarte a tripsina e deixar o prato na incubadora por 2-3 min. Adicionar meio de cultura pré-aquecido no prato e misturar o meio com as células. </li><li> Contar o número de células usando um hemocitômetro. Centrífuga de duas alíquotas de suspensões de células contendo 5 x 10 5 células por cada 5 min a 300 x g. Aspirar o sobrenadante completamente. </li><li> Depois de aspirar o sobrenadante completamente, ressuspender um sedimento dos pips sedimentos celulares em 50 ul de DM. Injetar a suspensão de células no lúmen do vaso descelularizados. </li><li> Em seguida, voltar a suspender o outro pellet pips celular em 100 ul de Matrigel. Pipeta cuidadosamente a mistura sobre o enxerto aorta descelulada. </li><li> Espere por 10-15 min até que a mistura se transforma em um estado de gel-like e uniformemente envolve em torno de superfície externa do navio. Encha a câmara de incubação com DM. </li><li> Colocar todo o biorreactor configuração em um 5% de CO2 a 37 ° C. Rodar manualmente o enxerto descelularizado 90 ° em torno do eixo longitudinal a cada meia hora durante as primeiras 2 horas. </li><li> Mantenha cultura estática durante 12 horas para permitir a adesão celular. </li><li> MS entregar através do lúmen pela bomba peristáltica para induzir a diferenciação de células de músculo liso. Iniciar a taxa de fluxo inicial de 5 ml / min e aumento gradual de 20 mL / min ao longo de 24 h. </li><li> Manter a taxa de fluxo do meio que circula em 20 ml / min durante 24 horas. </li><li> Interromper o fluxo médio e mover a configuração fora da incubadora biorreator. </li><li> Re-semente a luz do enxerto com PPCPE. Trypsinize, contar e centrífuga de 1 x 10 6 PPCPE como nos passos 5,5-5,6. Ressuspender as células em 50 ul de meio EGM-2 contendo 50 ng / ml de VEGF. Injectar a mistura de células para o lúmen do enxerto. </li><li> Alterar a forma da câmara de incubação e toda a circulação de meio EGM-2 que continha 50 ng de VEGF / mL para induzir a diferenciação endotelial. </li><li> Mova o biorreator configuração de volta ao the incubadora. Rodar manualmente o enxerto 90 ° de cada 30 min durante as primeiras 2 horas e, em seguida, manter uma cultura estática, durante 12 h. </li><li> Começar a circular o fluxo de 5 ml / min e aumento gradual de 35 ml / min. Manter a taxa de fluxo a 35 ml / min, durante 5 dias. Alterar circulando e médio câmara a cada dois dias. </li><li> Colher o enxerto de engenharia para posterior enxertia para mouse. </li></ol> 6. Alongamento das Duplo-semeado PPCPE Graft em Ratos <ol><li> Uso NOD.CB17-Prkdc scid / NcrCrl ratos macho como receptores de enxertos navio. Assegure-se sempre que os ratos são cerca de 10 semanas de idade, pesar cerca de 25 - 35 g, e estão em boas condições de saúde. </li><li> Anestesiar rato destinatário através da administração de uma combinação de Hypnorm (25 mg / kg) e Hypnovel (25 mg / kg) por via intraperitoneal. Aplique vaselina pomada oftálmica nos olhos para evitar a secura e sob anestesia. Confirme anesthetization eficiente, garantindo que os ratos têm músculos relaxados e está respirando de forma constante. <ol><li> Corrigir o mouse em decúbito dorsal com o seu pescoço raspada e estendida. Administrar sulfato de atropina (1,7 mg / kg) em combinação com os anestésicos para assegurar que o tracto respiratório do rato permanece claro. </li></ol></li><li> Prepare uma incisão na linha média da mandíbula para esterno do mouse. Sob um microscópio de dissecação com 5- a 10-vezes a amplificação, levantar as glândulas salivares direita lateralmente e remover o músculo cleidomastoid direito para expor a artéria carótida comum direita. </li><li> Suavemente remover tecidos ligados a mobilizar a artéria carótida comum direita a partir da extremidade distai para a extremidade proximal. Ligadura a porção média da artéria carótida comum, duas vezes com uma sutura de seda 8-0 e dissecar entre os dois laços. </li><li> Passe por uma braçadeira feita de tubo de nylon autoclavable em cada extremidade do vaso e corrigir cada extremidade com grampos microhemostat. </li><li> Remover laço de sutura numa extremidade e aplicar uma gota de heparina (100 U / ml). Imediatamente depois, inverter a extremidade distal do the artéria para cobrir todo o corpo cuff e corrigir o navio invertido com uma sutura 8-0 seda para o manguito. <ol><li> Repetir o mesmo procedimento para a outra porção da artéria. Lave artéria com solução salina para remover coágulos sanguíneos. </li></ol></li><li> Implantar um enxerto navio descelulada entre duas extremidades da artéria carótida, deslizando as extremidades dos vasos decelularizadas do ao longo dos punhos das artérias e fixando-o com fio de seda 8-0. Remover pinças vasculares a partir de qualquer termina antes de avaliar a pulsação do enxerto. </li><li> Coloque a glândula salivar de volta à sua posição original. Fechar a pele sobre o local cirúrgico com uma sutura interrompida utilizando um fio de sutura 6-0 de poliglactina. </li><li> Consistentemente observar os sinais vitais do rato após o procedimento for concluído, até que ele retoma a consciência. Sacrifique ratos receptores por luxação cervical e vasos colheita enxertos ou 24 horas ou 3 semanas mais tarde, para posterior análise. </li></ol>

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O protocolo atual indica um som, rápido, estratégia simples, eficiente e reprodutível em que os navios da engenharia de tecidos funcionais podem ser gerados usando PPCPE a partir de fibroblastos humanos. Esta técnica é uma ferramenta valiosa para a medicina regenerativa, engenharia de tecidos e terapia celular específico paciente potencialmente no futuro próximo. As etapas críticas para assegurar a eficácia do protocolo inclui a preparação de PPCPE, preparação de aórticos descelularizados totalmente estér...

A construção de condutas vasculares é uma estratégia fundamental para a reparação cirúrgica de vasos danificados e feridos resultantes de doenças cardiovasculares. Até à data, materiais de enxerto utilizados em cirurgia incluem biocompatível polímeros sintéticos (politetrafluoretileno [Teflon], politetrafluoretileno expandido [ePTFE; Gore-Tex] ou tereftalato de polietileno [Dacron]), enxertos, tecido autólogo (pericárdio ou veia safena) e xenotransplantes 1. Enquanto enxertos artificiais (por exemplo, Gore-Tex e Dacron) são mais comumente usados, estes materiais provável causar inúmeras complicações a curto e longo prazo que incluem estenose, a deposição de cálcio, trombo-embolia e infecções. Embora os pacientes com enxertos biológicos apresentam redução de eventos tromboembólicos, eles ainda se deparam com limitações, como a falência do enxerto secundário e durabilidade encurtado devido à calcificação degradação 2. Portanto, apesar das melhorias significativas na cirúrgico techniques longo dos anos, pesquisadores e clínicos ainda estão sobrecarregados com a necessidade de identificar a conduta ideal para doenças vasculares. Mais recentemente, no campo da engenharia de tecido vascular investigação tem gerado um conceito em que as células são incorporados em suportes biodegradáveis, com o objectivo de criar um ambiente biomimética que simboliza um recipiente funcional de um enxerto bem sucedido. Fundamentalmente, o sucesso das construções vasculares dependem de três componentes essenciais; As células que compreendem o andaime, ou seja, uma camada interior de células endoteliais e uma camada de células de músculo liso, um andaime que contém a matriz extracelular apropriado para proporcionar propriedades mecânicas comparáveis ​​à vasculatura nativa, e a sinalização celular / molecular que é necessário para iniciar / regulação reparação. Dos enxertos a longo prazo e desenvolvimento sustentado dos neo-tecidos são altamente dependentes de semeadura de células eficaz de andaimes, thereby tornar a decisão do tipo de célula de importância crítica. Vários relatos demonstram a utilização de células endoteliais maduras e células do músculo liso a partir de várias fontes para desenvolver condutas de diâmetro pequeno 3-6. Embora promissora, a falta de vasos autólogos suficientes para se obter endoteliais maduras e células musculares lisas continuam a ser um fardo considerável. Mais recentemente, as células estaminais a partir de várias fontes têm sido exploradas para aplicações de engenharia de tecidos vasculares. Células mononucleares de fato, uma grande variedade de tipos de células-tronco, incluindo células-tronco embrionárias 7, células estaminais pluripotentes induzidas (iPSCs) 8,9, PPCPE 10,11, derivadas da medula óssea 12, células-tronco mesenquimais 13, células progenitoras endoteliais e da parede do vaso adulto -derived célula estaminal antigénio-1 (Sca-1) + de células estaminais / progenitoras 14,15 têm sido demonstradas ser capazes de se diferenciarem em células endoteliais ou funcional ou de músculo liso, em resposta a meios definidos econdições de cultura. Além disso, a capacidade de auto-renovação ilimitada das células estaminais torná-los melhores candidatos ao contrário endoteliais maduras e as células musculares lisas, que apenas se podem dividir para um número limitado de vezes antes de se submeter a paragem do crescimento e senescência. A seleção do material andaime para gerar tecido sucesso navio projetado para enxertia depende de vários fatores, tais como biocompatibilidade, propriedades biomecânicas, e taxa de biodegradação. Fundamentalmente, os materiais usados ​​para criar suportes para os enxertos devem ser biodegradáveis ​​e não será montado receptores desnecessária respostas imunes. Além disso, ele deve abranger uma porosidade adequada e microestrutura para a fixação das células e subsequente sobrevivência. Até à data, os materiais mais comuns utilizados para suportes na engenharia de tecido vascular incluem polímeros de ácido poliglicólico, ácido poliláctico, e poli-caprolactona ε 16. Mais recentemente, materiais biológicos têm decelularizadastambém sido aplicada com algum sucesso. Vários laboratórios mostraram que a semeadura vasos porcino descelularizado humano, canino ou com células autólogas proporcionado um enxerto biológico que resistiu a coagulação e a hiperplasia intimal 17-19. Outras estratégias de engenharia de tecido vascular incluem enxertos vasculares extracelulares baseada em proteínas da matriz, por exemplo, células de semeadura em gel de fibrina 13 e folhas de células geradoras sem apoio andaime 20, 21. O actual protocolo demonstra a diferenciação de PPCPE humano em endotelial funcional e células do músculo liso, a geração de um biorreactor que consiste em um recipiente de andaime descelularizado para abrigar células vasculares PPCPE derivados funcionais, e enxerto de vasos em engenharia de tecidos de imunodeficiência combinada severa (SCID ) camundongos. PPCPE são um tipo de célula ideal para usar para engenharia de tecidos de enxertos de vasos porque essas células não formam tumores em camundongos ou aumentar ética eas respostas alo-imunes. Além disso, mostramos que a estratégia para a geração de células musculares pips-células endoteliais e pips-suave é eficiente e reprodutível 10,11. Depois disso, foi elaborado um navio descelulada para a semeadura de células vasculares PPCPE derivados para imitar de perto as proteínas da matriz que existe dentro de um vaso nativo, reforçando assim a enxertia e sobrevivência eficácia. Além disso, a descelularização dos vasos antes da semeadura PPCPE impede a ocorrência de reacções inflamatórias montados por tipos de células imunes tais como os macrófagos. Mais importante, este protocolo não representa apenas uma metodologia para gerar prometendo condutas vasculares para a tradução para os seres humanos, mas também fornece valioso meio de estudar e compreender os mecanismos moleculares que governam a regeneração do tecido vascular através de modelos de mouse.

<table><tbody><tr><td>Human Fibroblasts CCL-153</td><td>ATCC</td><td>CCL-153</td><td>Prenatal human embryonic fibroblasts</td></tr><tr><td>ATCC F-12K Medium (Kaighn&#039;s Modification of Ham&#039;s F-12 Medium)</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Knockout DMEM medium optimized for embryonic stem cells</td><td>Life technologies (Gibco)</td><td>12660-012</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Life technologies (Invitrogen)</td><td>10828-028</td><td></td></tr><tr><td>Human Basic FGF-2&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-837</td><td></td></tr><tr><td>alpha-MEM medium</td><td>Life technologies (Invitrogen)</td><td>32571093</td><td></td></tr><tr><td>Human PDGF</td><td>R&amp;amp;D System</td><td>120-HD-001</td><td></td></tr><tr><td>Gelatin Solution 2%</td><td>Sigma</td><td>G1393</td><td></td></tr><tr><td>Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td><td>Addgene</td><td>20866</td><td></td></tr><tr><td>&amp;nbsp;PvuI Restriction Enzyme</td><td>New England Biolabs</td><td>RO150S</td><td></td></tr><tr><td>SureClean Plus</td><td>Bioline</td><td>BIO-37047</td><td></td></tr><tr><td>Nucelofection Kit (NHDF Kit)</td><td>LONZA</td><td>VPD-1001</td><td></td></tr><tr><td>Neomycin</td><td>SIGMA</td><td>G418</td><td></td></tr><tr><td>KL 1500 LCD, Illumination for Stereo Microscopy</td><td>SCHOTT</td><td>KL 1500 LCD</td><td>Cold light illumination for stereo microscopy</td></tr><tr><td>Nikon Zoom Steromicroscope SMZ800</td><td>Nikon</td><td>SMZ800</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>10% SDS Stock Solution Molecular Biology Reagent</td><td>Severn Biotech</td><td>CAS 151-21-3</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Matrigel (10mg/ml)</td><td>BD</td><td>A6661</td><td></td></tr><tr><td>Shaker IKA Vibrax with Shaking platform VX 7&amp;nbsp;</td><td>Jepson Bolton&#039;s, Janke&amp;amp;Kunkel</td><td>S32-102</td><td></td></tr><tr><td>Masterflex L/S Digital Pump Drive</td><td>Cole-Parmer</td><td>WZ-07523-80</td><td></td></tr><tr><td>Masterflex L/S 6-channel, 6-roller cartridge pump head</td><td>Cole-Parmer</td><td>EW-07519-15</td><td></td></tr><tr><td>Masterflex L/S large cartridges for pump head</td><td>Cole-Parmer</td><td>EW-07519-75</td><td></td></tr><tr><td>Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td><td>Cole-Parmer&amp;nbsp;</td><td>WZ-96410-14</td><td>Tubing goes through the peristaltic pump</td></tr><tr><td>0.5mm ID, 0.8 mm OD Silicone Tubing</td><td>SILEX</td><td>N/A</td><td>Tubings connect incubation chamber, media reservoir and compliance chamber&amp;nbsp;</td></tr><tr><td>Fitting Reducer 0.5 to 1.6, natural Polypropyline</td><td>Ibidi</td><td>10829</td><td>Adapter connect above two types of tubings</td></tr><tr><td>1/32&quot; Tubing, ID 0.01&quot; (250&amp;micro;m) Material: PEEK</td><td>LabSmith</td><td>T-132-010P</td><td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td></tr><tr><td>One-Piece Fittings&amp;nbsp;</td><td>LabSmith</td><td>T-132-100</td><td>Fix the above tubings through the incubation chamber wall</td></tr><tr><td>Nylon tubes (OD 0.9mm, ID 0.75mm)&amp;nbsp;</td><td>Smiths Medical</td><td>N/A</td><td>Tubings insert into two ends of the aorta graft</td></tr><tr><td>NOD.CB17-Prkdc&lt;sup&gt;scid&lt;/sup&gt;/NcrCrl mouse</td><td>Charles River</td><td></td><td></td></tr><tr><td>Surgical sutures, 8-0&amp;nbsp; silk</td><td>ETHICON</td><td>W819</td><td></td></tr><tr><td>Hypnorm</td><td>Vetapharm</td><td>Vm21757/4000</td><td>Neuroleptanalgesic for use in mice</td></tr><tr><td>Hypnovel (Midazolam)</td><td>Roche</td><td>59467-70-8</td><td>Induction of anaesthesia</td></tr><tr><td>Dissecting microscope</td><td>Carl Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Nylon Tubing</td><td>Portex LTD</td><td>800/200/100/200</td><td>0.65 mm in diameter and 1 mm in length; to make artery cuff</td></tr><tr><td>Electrocoagulator</td><td>Martin&amp;nbsp;</td><td>SN 54.131</td><td>Ligation of artery branches on aorta</td></tr><tr><td>Bipolar micro hemostat forceps</td><td>Martin</td><td>80-91-12-04</td><td>Fixation of vessel ends</td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFX-7</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFL-3dZ</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>D-5aZ</td><td></td></tr><tr><td>Mini applier&amp;nbsp;</td><td>AESCULAP</td><td>FE572K</td><td></td></tr><tr><td>Micro hemostats clips</td><td>AESCULAP</td><td>FE720K</td><td></td></tr><tr><td>Surgical sutures, 6-0 VICRYL</td><td>ETHICON</td><td>V489</td><td></td></tr></tbody></table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

A geração bem sucedida de PPCPE foi confirmada 4 dias após nucleofecting fibroblastos humanos com um plasmídeo linearizado pCAG2LMKOSimO transportando quatro fatores de transcrição, Oct4, SOX2, KLF4 e c-Myc (OSKM). PPCPE apresentaram um fenótipo marcadamente distinta quando comparados com os fibroblastos (Figura 2A) e expressa os quatro factores de reprogramação no ARNm (Figura 2B) e de proteína (Figura 2C) 10 níveis. A eficácia de um enxerto vasc...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Aqui, apresentamos um protocolo para gerar engenharia de tecidos enxertos de vasos que são funcionais para enxerto em camundongos por semeadura dupla parcialmente célula-tronco pluripotentes induzidas (PPCPE) - derivados de células musculares lisas e PPCPE - células endoteliais derivadas em um biorreator descelulada navio andaime.

Geração e Alongamento das embarcações da engenharia de tecidos em um modelo de rato

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.9K visualizações.  King's College London BHF Centre.  Aqui, apresentamos um protocolo para gerar engenharia de tecidos enxertos de vasos que são funcionais para enxerto em camundongos por semeadura dupla parcialmente célula-tronco pluripotentes induzidas (PPCPE) - derivados de células musculares lisas e PPCPE - células endoteliais derivadas em um biorreator descelulada navio andaime. 

Vídeo: Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Transplante ortotópico de aorta em camundongos para o Estudo da Doença Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Medicina

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Os modelos de mouse para Arteriosclerose Graft

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Técnica Cirúrgica para a Implantação de engenharia de tecidos enxertos vasculares e Subsequente<em&gt; In Vivo</em&gt; Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Bioengenharia

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murino Cervical Modelo de coração Transplante usando uma técnica Cuff Modificado

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Implantação de Veia Cava Inferior interposição de enxerto no Rato Modelo

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

A escala do Engineered enxertos vasculares Usando 3D Impresso Guides and the Ring método de empilhamento

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

A Microsurgical Modelo Novel para heterotópico, em bloco da parede torácica, Thymus, e Transplante Cardíaco em Ratos

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

A propagação e a implantação de um enxerto traqueal biossintética engenharia de tecido em um modelo do rato

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Usando a técnica de manga em um modelo do rato da transplantação da aorta - um vídeo instrutivo

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Geração e Alongamento das embarcações da engenharia de tecidos em um modelo de rato

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