Name: generation and grafting of tissue-engineered vessels in a mouse model
Uploaded: 2015-03-18T14:00:00
Description: Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

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 substituiç?...

<ol>
	<li>Kurobe, H., Maxfield, M. W., Breuer, C. K., Shinoka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+Review:+Tissue-Engineered+Vascular+Grafts+for+Cardiac+Surgery:+Past,+Present,+and+Future.">Concise Review: Tissue-Engineered Vascular Grafts for Cardiac Surgery: Past, Present, and Future.</a> Stem Cells Transl Med. 1 (7), 566-571 (2012).</li><li>Jonas, R. A., Freed, M. D., Mayer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+follow-up+of+patients+with+synthetic+right+heart+conduits.">Long-term follow-up of patients with synthetic right heart conduits.</a> Circulation. 72, II77-II83 (1985).</li><li>Heureux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technology+insight:+the+evolution+of+tissue-engineered+vascular+grafts-from+research+to+clinical+practice.">Technology insight: the evolution of tissue-engineered vascular grafts-from research to clinical practice.</a> Nat Clin Pract Cardiovasc Med. 4, 389-395 (2007).</li><li>Zhang, W. J., Liu, W., Cui, L., Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+blood+vessel.">Tissue engineering of blood vessel.</a> J Cell Mol Med. 11, 945-957 (2007).</li><li>Cearbhaill, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+mesenchymal+stem+cells+to+the+biomechanical+environment+of+the+endothelium+on+a+flexible+tubular+silicone+substrate.">Response of mesenchymal stem cells to the biomechanical environment of the endothelium on a flexible tubular silicone substrate.</a> Biomaterials. 29, 1610-1619 (2008).</li><li>Gong, Z., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-diameter+human+vessel+wall+engineered+from+bone+marrow-derived+mesenchymal+stem+cells+(hMSCs).">Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs).</a> FASEB J. 22, 1635-1648 (2008).</li><li>Wong, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-expression+of+HSP47+augments+mouse+embryonic+stem+cell+smooth+muscle+differentiation+and+chemotaxis.">Over-expression of HSP47 augments mouse embryonic stem cell smooth muscle differentiation and chemotaxis.</a> PLoS One. 9 (1), e86118 (2014).</li><li>Park, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+differentiation+of+human+pluripotent+stem+cells+into+functional+CD34++progenitor+cells+by+combined+modulation+of+the+MEK/ERK+and+BMP4+signaling+pathways.">Efficient differentiation of human pluripotent stem cells into functional CD34+ progenitor cells by combined modulation of the MEK/ERK and BMP4 signaling pathways.</a> Blood. 116, 5762-5772 (2010).</li><li>Samuel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functionally+competent+and+durable+engineered+blood+vessels+from+human+induced+pluripotent+stem+cells.">Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells.</a> Proc Natl Acad Sci USA. 110, 12774-12779 (2013).</li><li>Margariti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+fibroblasts+into+endothelial+cells+capacble+of+angiogenesis+and+reendothelialization+in+tissue-engineered+vessels.">Reprogramming of fibroblasts into endothelial cells capacble of angiogenesis and reendothelialization in tissue-engineered vessels.</a> Proc Natl Acad Sci USA. 109, 13793-13798 (2012).</li><li>Karamariti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+cells+differentiated+from+reprogrammed+embryonic+lung+fibroblasts+through+DKK3+signaling+are+potent+for+tissue+engineering+of+vascular+grafts.">Smooth muscle cells differentiated from reprogrammed embryonic lung fibroblasts through DKK3 signaling are potent for tissue engineering of vascular grafts.</a> Circ Res. 112, 1433-1443 (2013).</li><li>Udelsman, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+operator-independent+method+for+seeding+tissue-engineered+vascular+grafts.">Development of an operator-independent method for seeding tissue-engineered vascular grafts.</a> Tissue Eng Part C Methods. 17 (7), 731-736 (2011).</li><li>Cearbhaill, E. D., Murphy, M., Barry, F., McHugh, P. E., Barron, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+human+mesenchymal+stem+cells+in+fibrin-based+vascular+tissue+engineering+constructs.">Behavior of human mesenchymal stem cells in fibrin-based vascular tissue engineering constructs.</a> Ann Biomed Eng. 38 (3), 649-657 (2010).</li><li>Wong, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+control+vascular+stem/progenitor+cell+plasticity+through+tumor+necrosis+factor-&#945;-mediated+nuclear+factor-&#954;B+activation.">Macrophages control vascular stem/progenitor cell plasticity through tumor necrosis factor-&#945;-mediated nuclear factor-&#954;B activation.</a> Arterioscler Thromb Vasc Biol. 34 (3), 635-643 (2014).</li><li>Wong, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirolimus+stimulates+vascular+stem/progenitor+cell+migration+and+differentiation+into+smooth+muscle+cells+via+epidermal+growth+factor+receptor/extracellular+signal-regulated+kinase/&#946;-catenin+signaling+pathway.">Sirolimus stimulates vascular stem/progenitor cell migration and differentiation into smooth muscle cells via epidermal growth factor receptor/extracellular signal-regulated kinase/&#946;-catenin signaling pathway.</a> Arterioscler Thromb Vasc Biol. 33 (10), 2397-2406 (2013).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+matrices:+State+of+the+art.">Three-dimensional cell culture matrices: State of the art.</a> Tissue Eng Part B Rev. 14, 61-86 (2008).</li><li>Hung, H. S., Hsu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Advances+of+stem+cell-based+approaches+to+tissue-engineering+vascular+grafts.">Current Advances of stem cell-based approaches to tissue-engineering vascular grafts.</a> OA Tissue Engineering. 1 (1), 2 (2013).</li><li>Quint, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tissue-engineered+blood+vessel+as+an+arterial+conduit.">Decellularized tissue-engineered blood vessel as an arterial conduit.</a> Proc Natl Acad Sci USA. 108 (22), 9214-9219 (2011).</li><li>Zhang, X., Xu, Y., Thomas, V., Bellis, S. L., Vohra, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+an+antiplatelet+adhesion+layer+on+an+electrospun+scaffold+using+porcine+endothelial+progenitor+cells.">Engineering an antiplatelet adhesion layer on an electrospun scaffold using porcine endothelial progenitor cells.</a> J Biomed Mater Res A. 97 (2), 145-151 (2011).</li><li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+use+of+an+induced+puripotent+stem+cell+sheet+for+the+construction+of+tissue-engineered+vascular+grafts.">Evaluation of the use of an induced puripotent stem cell sheet for the construction of tissue-engineered vascular grafts.</a> J Thorac Cardiovasc Surg. 143 (3), 696-703 (2012).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+to+engineer+small-diameter+vascular+grafts+from+marrow-derived+mesenchymal+stem+cells.">A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesenchymal stem cells.</a> Artif Organs. 36 (1), 93-101 (2012).</li><li>Tsai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+stem+cells+to+neointimal+formation+of+decellularized+vessel+grafts+in+a+novel+mouse+model.">Contribution of stem cells to neointimal formation of decellularized vessel grafts in a novel mouse model.</a> Am J Pathol. 181 (1), 362-373 (2012).</li><li>Kasimir, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+decellularization+procedures+of+porcine+heart+valves.">Comparison of different decellularization procedures of porcine heart valves.</a> Int J Artif Organs. 26 (5), 421-427 (2003).</li><li>Stephenson, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+propagation+of+human+embryonic+stem+cell+lines+from+frozen+embryos+in+an+animal+product-free+environment.">Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment.</a> Nature Protocols. 7, 1366-1381 (2012).</li><li>Takahashi, K., Okita, K., Nakagawa, M., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+fibroblast+cultures.">Induction of pluripotent stem cells from fibroblast cultures.</a> Nature Protocols. 2, 3081-3089 (2007).</li><li>McCall, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+and+intramyocardial+injection+models+in+swine.">Myocardial infarction and intramyocardial injection models in swine.</a> Nature Protocols. 7, 1479-1496 (2012).</li><li>Olausson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+an+allogeneic+vein+bioengineered+with+autologous+stem+cells:+a+proof-of-concept+study.">Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study.</a> Lancet. 380 (9838), 230-237 (2012).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" >
	<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&#39;s Modification of Ham&#39;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&#160;</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&#38;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 >&#160;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>Selection of&#160;</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&#39;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&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;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&#160;</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&#160;</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&#34; Tubing, ID 0.01&#34; (250&#181;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&#160;</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)&#160;</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-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; 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&#160;</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&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</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. ...

Research

JoVE Journal

Bioengineering

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Protocolo Relativo Avaliação hidrodinâmica de Tri-folheto polímero Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Bioengenharia

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Preparação de Membranas de Luz respondem por um enxerto de superfície combinada e Processo Postmodification

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

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

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

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Tissue Combinada 3D Engineered<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Modelo de tumor de pulmão para prever a eficácia da droga em fundos específicos de mutação

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

aplicações de<em&gt; In Vivo</em&gt; Teste Funcional do Rato tibial anterior para a Avaliação da engenharia de tecidos Repair Músculo Esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Engenharia de Tecido Tridimensional de Redes de Astrócitos Alinhadas para Recapitular Mecanismos de Desenvolvimento e Facilitar a Regeneração do Sistema Nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Uma nova técnica de sutura tenorrhaphy com enxerto de colágeno projetado por tecido para reparar defeitos grandes do tendão

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

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

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 ...