D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. &#160;Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

Todos os procedimentos são realizados à temperatura ambiente (~ 23 ° C) a menos que indicado de outra forma. 1. Fabricação de Silicon Mestre <ol><li> Pastilha de silício Limpe com etanol ou xileno e seco de 100% em O 2 / N 2 gás. </li><li> Coloque bolacha de silício em spin-coater a velocidades de rotação de 2.000-4.000 rpm para produzir um filme de espessura de 0,3-0,5 um. </li><li> Padrão do filme foto-resistente com as dimensões correctas, usando um sistema de fotolitografia </li><li> Totalmente mergulhar as pastilhas de silício revestido de fotossensíveis estampados em um volume adequado de solução photoresist desenvolvimento. </li><li> Lavar as lâminas de silício revestido de fotossensíveis desenvolvidos com água deionizada. </li><li> Para formar matrizes de sub-mícron cumes escala com perto de paredes laterais verticais, profundo etch iônica reativa o silício exposto usando um sistema de ataque. </li><li> Remover o fotorresistente remanescente colocando a bolacha de silício em um sistema de incinerador do plasma. </li><li> Corte o silício wAfers com um cortador em mestres de silício dimensionadas adequadamente com ponta de diamante para moldagem réplica posterior. </li></ol> 2. Fabricação de PUA molde de silicone Mestre Nota: O volume a ser adicionado ao mestre de silício para nanofabrication irá variar, dependendo da área do mestre nanopatterned a ser replicado como também a viscosidade da solução (PUA) acrilato de poliuretano. <ol><li> Limpe a superfície mestre de silício com 100% de etanol ou xileno e seco sob O 2 / N 2 gás. </li><li> Coloque padrão mestre de silício lado em uma placa de Petri. </li><li> Para um mestre de silício com de 2 cm x 2 cm da superfície do teste padrão, pipeta 40 ul de PUA para a superfície do padrão. </li><li> Colocar uma folha de 4 cm x 4 cm transparente de poliéster (PET) de película sobre o PUA dispensado. </li><li> Pressione para baixo sobre a folha de PET e espalhar o PUA debaixo da folha em toda a face padrão, usando um rolo ou de superfície plana com arestas (tal como uma placa), de modo que a totalidadepadrão é coberto pelo pré-polímero PUA. </li><li> Coloque mestre de silício, pré-polímero, e PET aproximadamente 10 cm abaixo de uma luz de 20 watts (115 V) UV (λ = 365 nm) por 50 segundos. Para ser eficaz, o comprimento de onda de luz ultravioleta pode ser em qualquer lugar entre 310-400 nm. A intensidade da luz é de 10-15 mW / cm 2, na superfície do substrato. </li><li> Após a cura, remova filme PET lentamente com uma pinça. PUA deve anexar ao filme PET com uma negativa do mestre nanopattern silício. </li><li> Cure nanopatterns PUA / PET sob UV, durante pelo menos 12 horas antes de usar. Superexposição não é um problema. </li><li> Para limpar mestres de silício, coloque outro filme de PET em cima do mestre, sem a adição de PUA e expor à luz UV (λ = 365 nm) por 50 segundos e retire películas PET. Isto irá remover todos os monômeros não reagidos. </li><li> Enxágüe mestre de silício com 100% de etanol ou xileno e seco sob O 2 / N 2 gás. </li></ol> 3-A. Microusinagem poliuretano Polímero </p&gt; <ol><li> Preparar 25 milímetros de diâmetro lâminas de vidro circulares, introduzindo num compartimento de tratamento de ozono durante 10 min. </li><li> Coloque lâminas de vidro tratadas com ozônio em pequeno bloco PDMS para fácil manuseio. </li><li> Aplicar fina camada de aderência promotor superfície com pincel para lâminas de vidro. Ar lâminas de vidro seco durante 30 min. </li><li> Coloque a lâmina de vidro em um pedaço de papel da impressora. </li><li> Largue dispensar 10 ml de poliuretano (PU) pré-polímero (NOA 76) para o centro da lâmina de vidro. Certifique-se sem bolhas estão presentes após a adição. </li><li> Molde Lugar PUA, padrão de face para baixo, sobre a lâmina de vidro. Dispersar o PU uniformemente em toda a superfície da lâmina de vidro rolando um rolo de cilindro de borracha ao longo do molde PUA. Papel para impressora irá absorver excesso de polímero. </li><li> Lâmpada UV Watt. Tempo de polimerização do PU é dependente da potência da fonte de UV. </li><li> Retirar a amostra da fonte de luz UV e retire com cuidado o molde PUA de PU revestido lâmina de vidro. Polimerização do PU é considerado complete quando as cascas de molde PUA limpa distância a partir da amostra e a lamela de vidro de PU tem uma aparência iridiscente. </li><li> Coloque amostras terminados em dessecador para o armazenamento durante o tempo que um mês. </li></ol> 3b. Microusinagem Poli (láctido-co-glicólido) Hidrogel <ol><li> Criar um molde PDMS plana por vigorosa mistura silicone base de elastômero de silicone e agente de cura de elastômero em uma proporção de 10:1. </li><li> Despeje PDMS mistos solução precursora em uma placa de Petri de modo a que o precursor de PDMS atingir 5 mm até a borda do prato (isto é, de modo que o PDMS tem 5 mm de espessura). </li><li> Colocar uma placa de Petri e PDMS precursor num exsicador durante 1 hora para desgaseificar. </li><li> Mover uma placa de Petri e PDMS precursor num forno a 65 ° C durante pelo menos 2 horas para curar. </li><li> Depois de PDMS é curado, use uma navalha para cortar os PDMS planas em três centímetros x 3 cm seções quadrados. Estes serão os PDMS planas moldes utilizados mais tarde no processo de modelação. </li><li> Limpo 25 mm de diâmetro lâminas de vidro circular por plrastreio em álcool isopropílico, durante 30 min num sonicador de água. </li><li> Lâminas de vidro limpo a seco com menos de O 2 / N 2 gás. </li><li> Gota dispensar 100 ul de uma solução de PLGA (15% w / v de PLGA em clorofórmio) na lâmina de vidro. </li><li> Coloque um molde PDMS plana no topo do PLGA dispensado para absorver o solvente e obter uma superfície plana de PLGA. Aplicar uma ligeira pressão (~ 10 kPa), colocando um peso de 200 g na parte superior do PDMS de 5 min. </li><li> Lentamente descascar molde PDMS plana e colocar a tampa de vidro sobre um prato pré-aquecido (120 ° C) durante 5 minutos para remover o solvente residual e aumentar a aderência entre o PLGA e o vidro de cobertura. </li><li> Coloque o molde NP PUA no topo do PLGA plana e aplicar pressão constante (~ 100 kPa) e de calor (120 ° C) colocando um peso de 1 kg, em cima do molde de PUA, enquanto na placa de aquecimento durante 15 min. </li><li> Remover o peso de PLGA a tampa deslizante e permitir que o substrato arrefecer até à temperatura ambiente. Não retire o molde PUA até os substratos foramarrefecida. </li><li> Uma vez que os substratos tenham esfriado o suficiente, cuidadosamente descascar o molde NP PUA, revelando o substrato NP PLGA. O substrato NP PLGA deve ter uma aparência iridescente. </li><li> Coloque amostras terminados em dessecador para o armazenamento durante o tempo que um mês. </li></ol> 4. Sementeira e Cultura de Células NOTA: Este protocolo descreve a cultura de miócitos ventriculares de rato neonatal (NRVMs) e derivados de células humanas cardiomiócitos H7 estaminais embrionárias (hESC-MC), mas outras fontes de células podem ser utilizados. <ol><li> Anexar as lamelas ANFS (PU ou PLGA) para de 35 mm de cultura de tecidos de poliestireno prato. Pipetar 20 l de Norland Optical Adhesive (NOA83H) para o fundo do prato e colocar suavemente a lamela ANFS no topo da NOA. Permitir cola para se espalhar e cobrir todo fundo lamela. Cure NOA expondo prato a UV por 10 min. </li><li> Esterilizar ANFS lavando com 2 ml de solução aquosa de etanol a 70% durante 5 min, duas vezes. Remover eThanol por aspiração. Permitir ANFS para secar completamente ar por ~ 1 hora sob a esterilização lâmpada UV (λ = 200-290 nm) na cabine de segurança biológica. </li><li> Adesão celular é aumentada por revestimento do ANFS em fibronectina durante a noite. Dilui-se a fibronectina em água Dl a 5 ug / ml. Pipetar 2 ml da solução de fibronectina no prato. Colocar na incubadora a 37 ° C e 5% de CO 2 durante a noite (pelo menos 6 horas). </li><li> Obter NRVMs, hESC-CMs, ou outras células cardíacas de juros de acordo com os protocolos anteriores 22. </li><li> Centrífuga amostra de células a 1000 rpm durante 3 min para sedimentar as células. </li><li> Remova cuidadosamente o sobrenadante por aspiração. Certifique-se de não perturbar o sedimento. </li><li> Ressuspender as células em meio de cultura adequado a uma concentração de 4,6 x 10 6 células / ml. </li><li> Pipetar cuidadosamente 200 mL de suspensão de células para ANFS esterilizados. Certifique-se de suspensão de células permanece na lamela. </li><li> Colocar as células em estufa a 37 ° C e 5%CO 2 para 4 horas para permitir que as células para anexar ao ANFS. </li><li> Adicionar 2 ml de meio de cultura quente para prato e substituir as células em estufa nas mesmas condições. </li><li> Após 24 h, retirar o suporte e lava-se com 2 mL de DPBS duas vezes para remover o excesso de células. </li><li> Adicionar 2 ml de meio de cultura quente para prato e substituir as células em estufa nas mesmas condições. Cultura as células a confluência. Substituir mídia todos os dias. </li></ol>

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D., Bub, G., Lakireddy, P., El-Sherif, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+arrhythmias+in+the+cardiomyocyte+monolayer.">Optical imaging of arrhythmias in the cardiomyocyte monolayer.</a> Heart Rhythm. 9, 2077-2082 (2012).</li><li>Kim, J., Hayward, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+dynamic+in+vivo+environments+with+stimuli-responsive+materials+for+cell+culture.">Mimicking dynamic in vivo environments with stimuli-responsive materials for cell culture.</a> Trends in Biotechnology. 30, 426-439 (2012).</li><li>Henderson, D. J., Anderson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+and+Structure+of+the+Ventricles+in+the+Human+Heart.">The Development and Structure of the Ventricles in the Human Heart.</a> Pediatr Cardiol. 30, 588-596 (2009).</li><li>Badie, N., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Micropatterned+Cardiac+Cell+Cultures+with+Realistic+Ventricular+Microstructure.">Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure.</a> Biophysj. 96, 3873-3885 (2009).</li><li>Badrossamay, M. R., McIlwee, H. A., Goss, J. A., Parker, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+Assembly+by+Rotary+Jet-Spinning.">Nanofiber Assembly by Rotary Jet-Spinning.</a> Nano Lett. 10, 2257-2261 (2010).</li><li>Rao, 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=The+effect+of+microgrooved+culture+substrates+on+calcium+cycling+of+cardiac+myocytes+derived+from+human+induced+pluripotent+stem+cells.">The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells.</a> Biomaterials. 34, 2399-2411 (2013).</li></ol>

Authors have nothing to disclose.

Tecidos cardíacos funcionalmente maduros faltam para tanto in vivo e in vitro em aplicações de engenharia de tecido cardíaco. Os métodos de nanofabricação CFL aqui descritas são técnicas robustas para alcançar o alinhamento celular e influenciando a função do tecido macroscópico devido à escalabilidade do sistema. Grandes áreas podem ser facilmente modelado e usado para a cultura de células. Alinhamento celular macroscópica é essencial em engenharia de tecido cardíaco, a fim de criar...

A doença cardiovascular é a principal causa de morbidade e mortalidade no mundo e apresentar um peso sócio-econômico importante em uma 1,17 já tenso sistema de saúde global. Engenharia de tecidos cardíacos tem dois objetivos distintos: (1) para regenerar miocárdio danificado após doença isquêmica ou cardiomiopatia ou (2) para a criação de um modelo de alta fidelidade do coração para a triagem de drogas in vitro ou modelagem doença. O coração é um órgão complexo que deve trabalhar constantemente para fornecer sangue para o corpo. Estruturas laminares densamente embalado de cardiomiócitos e tecidos de suporte são dispostos em padrões helicoidais em toda a parede do coração 18,19. O coração também é eletromecanicamente acoplada 20 em uma forma altamente coordenada para ejetar o sangue eficientemente para o corpo 21. Vários grandes obstáculos ainda precisam ser abordados, no entanto, antes de desenho intrincado da natureza pode ser reproduzido de forma confiável in vitro.Em primeiro lugar, embora os métodos de diferenciação de cardiomiócitos robustas continuam a ser desenvolvidos 22, HPSC-CMs ainda exibem fenótipos bastante imaturos. Suas propriedades eletromecânicas e morfologia mais se aproximam os níveis fetais 23. Em segundo lugar, quando mantidos em condições de cultivo tradicionais, tanto com células-tronco derivadas e cardiomiócitos primários não conseguem montar em, estruturas de tecido-como nativas. Pelo contrário, as células tornam-se orientadas aleatoriamente e não apresentam a aparência em forma de haste em faixas de miocárdio adulto 24. O ambiente da matriz extracelular (ECM) com os quais interagem as células desempenha um papel importante em numerosos processos celulares 11,13,25. A ECM é composto por sinais moleculares e topográficos complexos, bem definidas, que influenciam de forma significativa a estrutura e função das células de 6,26. Dentro do coração, o alinhamento celular segue de perto as fibras ECM escala nanométrica subjacente 2. O impacto destes nanotopographpistas icas em células e a função do tecido, no entanto, está longe de ser completamente entendido. Estudos preliminares de interação célula-biomaterial escala nanométrica indicam a potencial importância eo impacto da sub-mícron sugestões topográficas para sinalização celular 27, adesão 28-30, crescimento de 31, e de diferenciação 32,33. No entanto, devido à dificuldade no desenvolvimento de substratos nanofabricated reprodutíveis e expansíveis, tais estudos podem não reproduzir os efeitos celulares multi-escala do complexo em ambiente de ECM in vivo. Neste protocolo, uma técnica de nanofabricação simples e de baixo custo para a produção de scaffolds de cultura celular que imita fibra de alinhamento ECM cardíaca nativa é descrito, permitindo uma ampla gama de novas investigações de interações cardiomiócitos-biomaterial. Entender como cardiomiócitos interagir com o ambiente ECM nanoescala pode permitir a capacidade de controlar o comportamento celular para mais de perto imitar func tecido nativoção. Além disso, as monocamadas de células são um sistema experimental simplificado em comparação com estruturas 3D, mas ainda apresentam um comportamento multicelular complexo para investigações criteriosas e triagem funcional 2,34-36. Finalmente, tais suportes pode ser usada para melhorar a função do enxerto celular quando implantado no coração para efeitos de regeneração 37.

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			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Plasma Asher System</td>
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			<td class="xl65" style="width:66pt" width="88">372978-1L</td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">500g Weights</td>
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			<td class="xl65" style="width:66pt" width="88">T9FB503120</td>
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capillary force lithography for cardiac tissue engineering

A doença cardiovascular continua a ser a principal causa de morte em todo o mundo 1. Engenharia de tecidos cardíacos muito promissora para entregar descobertas médicas inovadoras com os objectivos de desenvolvimento de tecidos funcionais para regeneração cardíaca, bem como ensaios in vitro de triagem. No entanto, a capacidade de criar modelos de alta fidelidade de tecido cardíaco tem sido difícil. Matriz extracelular do coração (ECM) é uma estrutura complexa composta de ambos os sinais bioquímicos e biomecânicos que variam a partir do micro-à escala nanométrica 2. Local condições de carga mecânica e interações célula-ECM foram recentemente reconhecidos como componentes vitais em engenharia de tecido cardíaco 3-5. Uma grande parte do ECM cardíaco é composto por fibras de colágeno alinhados com diâmetros em escala nano que influenciam significativamente a arquitetura do tecido e acoplamento eletromecânico 2. Infelizmente, alguns métodos have foram capazes de imitar a organização das fibras de ECM para baixo para a escala nanométrica. Os recentes avanços em técnicas de nanofabricação, no entanto, permitiram o projeto e fabricação de andaimes escaláveis ​​que imitam os sinais in vivo de rigidez estrutural e substrato do ECM no coração 6-9. Aqui apresentamos o desenvolvimento de duas reprodutíveis, os processos de microusinagem escaláveis ​​para o alinhamento funcional das células cardíacas usando o poli biocompatível polímero (ácido lático-co-glicólico) (PLGA) 8 e um poliuretano (PU) de polímero a base de custo-benefício, e. Estes substratos anisotropicamente nanofabricated (ANFS) imitar o ECM subjacente bem organizados, tecidos alinhados e pode ser utilizado para investigar o papel de nanotopografia na morfologia e função das células 10-14. Usando um mestre de silício nanopatterned (NP), como um modelo, um acrilato de poliuretano (PUA) molde é fabricado. Este molde PUA é então utilizado para pattern o hidrogel PU ou PLGA via assistida por UV ou litografia força capilar mediada por solvente (CFL), respectivamente 15,16. Resumidamente, PU ou PLGA de pré-polímero é gota dispensada para uma lamela de vidro e o molde PUA é colocada em cima. Para CFL assistida por UV, a UP é então exposto à radiação ultravioleta (λ = 250-400 nm) para a cura. Para mediada por solvente CFL, o PLGA é gravado utilizando calor (120 ° C) e pressão (100 kPa). Após a cura, o molde PUA é retirado, deixando para trás um ANFS para cultura de células. Pilhas, como miócitos ventriculares de ratos neonatos, bem como cardiomiócitos derivados de células-tronco pluripotentes humanas, pode ser mantido no ANFS 2.

A Figura 1 é uma vista geral esquemática do processo de produção para os dois métodos de fabrico. Devido à difracção da luz causada pela topografia nanoescala, microusinagem deve resultar numa superfície iridescente ao ANFS. Figura 2 representa essa superfície iridescente num bem modelado 25 milímetros NP-PU lamela (Figura 2A) com rebordo 800 nm e ranhura largura (Figura 2B). A aparência iridescente do ANFS vai variar um pouco dependendo do ...

Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

Capillary Force Lithography for Cardiac Tissue Engineering

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

capillary-force-lithography-for-cardiac-tissue-engineering

Research

JoVE Journal

Bioengineering

12.3K visualizações.  University of Washington. In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Vídeo: Capillary Force Lithography for Cardiac Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Biologicamente fabricação de materiais derivados injetável para Engenharia de Tecidos do Miocárdio

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Bioengenharia

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico Andaimes em Engenharia de Tecidos Arterial

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Padronização de plasma de superfície litografia para a Criação de Redes de celular

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Um protocolo para Decellularizing Cochleae Mouse para engenharia de tecidos do ouvido interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Methods Article

Capilar Força Litografia de Engenharia tecido cardíaco