Name: neonatal cardiac scaffolds: novel matrices for regenerative studies
Uploaded: 2016-11-05T14:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

The authors gratefully acknowledge Ms. Cynthia DeKay for the preparation of the figures.

Todas as experiências de rato foram realizados de acordo com o Animal Welfare Act dos EUA e foram aprovados pelo Comitê de Cuidado e Uso Institucional Animal da Universidade de Minnesota. 1. Método para coração mouse Isolamento <ol><li> Euthanize um rato neonatal por decapitação com uma única lâmina de uso. </li><li> Pincelar o tórax com 70% de etanol. </li><li> Dissecar a pele do peito, cortando-o afastado da parede torácica com uma tesoura padrão enquanto puxa a pele lateralmente com um par de # 5 fórceps. </li><li> Perfurar o abdômen logo abaixo do esterno com a tesoura de corte através da parede abdominal. Agarrando o processo xifóide com # 5 fórceps, retrair o esterno rostral a partir do corpo durante o corte que as nervuras de ambos os lados da caixa com a tesoura. A caixa torácica é refletida superiormente com a pinça para revelar o coração. </li><li> Sem rodeios dissecar os dois principais lóbulos do timo puxando lateralmente com # 5 fórceps, expondoo arco da aorta, bem como as veias cavas e pulmonares. </li><li> Transecto as principais artérias do arco aórtico e da própria aorta, com a tesoura primavera 10 cm. Reter a aorta entre a base do coração e a artéria inominada. Segure as extremidades dos vasos rompidos com o # 5 fórceps para refletir o coração para a frente, separando-a da traquéia e esôfago. </li><li> Transecto a vasculatura pulmonar e outras veias principais, através do corte entre os pulmões e o coração em ambos os lados do coração com a tesoura de mola 10 cm. As veias permanecer aberta para fornecer drenagem. </li><li> Tiraram o coração do mediastino com o # 5 fórceps, segurando as extremidades cortadas dos grandes vasos. Colocar o coração num prato de cultura de 60 mm contendo solução salina tamponada com fosfato estéril (PBS) para a cateterização. </li></ol> 2. Método para Decelularização por Langendorff Perfusão <ol><li> Prepare o conjunto de cateter com antecedência. Desenhar uma secção 4 centímetrosde PE 50 tubos em uma pequena chama de álcool para criar um cateter menores, mais finos. Aparar as extremidades com uma lâmina para satisfazer as dimensões (aprox. OD 300 mm de diâmetro exterior com uma flange de aprox. OD 500 mm) mostrado na Figura 1. Isto irá proporcionar dois cateteres simétricos de ambos os lados do puxar. </li><li> O ponto final do corte do tubo para dentro da chama brevemente, para derreter uma flange para a abertura na extremidade mais fina. </li><li> Encher a seringa de 12 ml com PBS e montar o cateter, colocando uma torneira de passagem de 3 vias em a seringa. Adicionar um 22 G x 1 agulha para a torneira e conduzir a agulha através do septo. Deslize o tubo do cateter PE arrastado para cima da agulha (fig. 1). </li><li> Lavar as peças montadas com PBS, assegurando que todas as bolhas de ar foram removidas. </li><li> Insira o cateter, preparado como descrito acima, para a aorta do coração isolado, não se estendendo para além da válvula aórtica e ligar com um empate de 7-0 sutura. Coloque o flange de tele cateter proximal contra o empate, fazendo um selo apertado e impedindo o coração de saindo do cateter. </li><li> Observe o cateterismo sob ampliação, enquanto suavemente perfusão do coração utilizando a seringa contendo PBS. Certifique-se de que não há vazamentos no sistema e o tecido homogeneamente empalidece como sangue latente é removido. </li><li> Colocar o septo para dentro do gargalo do adaptador para entrada e selá-lo, dobrando os lados sobre o vidro. </li><li> Anexar o reservatório cheio com 60 ml de sulfato de dodecilo de sódio a 1% (SDS) em água destilada (dH2O) através de uma linha de comprimento suficiente para produzir uma coluna que gera 20 mmHg de pressão, como mostrado na Fig. 1. Calcula-se a pressão da altura da coluna de líquido com base na relação de 1 mm Hg é igual a 1,3595 centímetros H 2 O. Cuidado: SDS é um pó altamente flocculent com propriedades irritantes. O contacto deve ser evitado. Lidar com o pó usando vestuário de protecção adequado. </li><li> Perfundir o coração com 1% SDS, durante 14 horas. O coração será translúcido na aparência sem tecido restante observável. </li><li> Lave o sistema até a torneira da solução SDS restante e substituí-lo com 10 ml dH 2 O. Perfundir o coração com 10 ml de 1% de Triton X-100 (diluídos em água destilada), seguido de 10 ml de dH 2 O, seguido por 60 ml de PBS contendo 1x penicilina estreptomicina (Pen-Strep). </li><li> Armazenar o coração em PBS 1x com Pen-Strep a 4 ° C. Se a aplicação de pós descelularização destina a perfusão requer, então, deve ser mantido o cateter na aorta, de outra forma reduzir o laço de sutura e o remover do cateter. </li></ol> Determinação 3. DNA <ol><li> Prepara-se uma digestão de ECM o coração ou o controlo descelularizado utilizando 200 ug / ml de proteinase K em KCl 50 mM, MgCl2 2,5 mM, 0,45% de Tween 20 em 10 mM de Tris (pH 8,3). </li><li> Incubar o tecido a 55 ° C com agitação até que o tecido é dissolvido,tipicamente 4-5 h. </li><li> Quantificar o teor de ADN do homogeneizado com um ADN de ensaio de ligação de 4,11. </li></ol> 4. Fixação e secção de tecido <ol><li> Fix descelularizados, corações repovoados ou P3 controlo em paraformaldeído a 4% em PBS durante 30 min à temperatura ambiente. </li><li> Lavar o tecido 3x em PBS. </li><li> Colocar o tecido numa solução de 7,5% de sacarose em tampão de fosfato 0,1 M a 4 ° C até que se afunda. </li><li> Mudar a solução a 15% de sacarose em tampão de fosfato 0,1 M a 4 ° C, novamente, até que se afunda. </li><li> Aquecer o tecido a 37 ° C, substituir metade do volume com solução de gelatina em 15% de sacarose e tampão de fosfato 0,1 M e equilibrar durante a noite. Concentrações de gelatina são aumentadas por passos através de 1%, 2,5%, 5% e, finalmente, 7,5%, duas vezes. </li><li> Coloque as amostras em cryomolds usando fresco gelatina 7,5%. Para restaurar o formato das amostras descelularizados, gelatina líquido pode ser perfundida para dentro das câmaras como tele corações são moldados. Congelar, flutuando os cryomolds em nitrogênio líquido. Armazenar a -80 ° C. </li><li> Cut (10 mm de espessura) seções com um criostato. Trazer o slide perto da superfície da seção e observe a seção de se mover para fora da faca na superfície devido à carga nos slides eletrostaticamente tratados. Secam-se as lâminas durante a noite e armazenar a -80 ° C para análise futura. </li><li> Retirar as lâminas do congelador, equilibrar à temperatura ambiente e, em seguida, colocar num frasco Coplin contendo PBS durante 20 min a 37 ° C para dissolver a gelatina. </li><li> Manchar as seções de corte a partir do Passo 4.7 com Hematoxilina e Eosina. Colocar as lâminas em um frasco Coplin. Expor as lâminas hidratados no passo 4.8 a hematoxilina durante 45 segundos, a água da torneira durante 1,5 min, tampão durante 3 min, a água da torneira durante 1 min, agente anil durante 1,5 min, 80% de etanol durante 1 min, alcoólica eosina Y durante 8 segundos, etanol a 80% durante 1 minuto, 100% de etanol durante 1 min 2x, agente de compensação (substituto de xileno) 3x durante 1 min, a lamela com resinameio de montagem com base. Examine os slides microscopicamente. </li><li> Para confirmar a retenção de reactividade proteína da MEC do ECM coração, mancha para proteínas estruturais de ECM tais como colagénio IV, colocando os diapositivos hidratadas numa câmara humidif içada e trata-se com 10% de soro de burro normal (NDS) em salino tamponado com fosfato com 0,1% de Triton -X100 (PBST) durante 1 h à temperatura ambiente (TA). Utilizar um volume suficiente para cobrir as secções de tecido. </li><li> Substituir a solução com o anticorpo primário de escolha para uma diluição determinada empiricamente em 5% NDS / PBST. Por exemplo, o anticorpo do colagénio IV foi diluído a 1: 150. Incubar durante a noite a 4 ° C. </li><li> Lavam-se as lâminas com PBST 3x e aplicar um anticorpo secundário de escolha conjugado com um corante fluorescente. Dilui-se o anticorpo por uma quantidade determinada empiricamente. Incubar durante 1 h à TA. Lavar 3x com PBS. </li><li> Corar as lâminas com um corante de ligação a ADN, tais como DAPI para verificar a ausência de núcleos celulares usando uma mounti contendo DAPIng médio quando a tampa deslizando os slides. </li><li> Examinar as lâminas com um microscópio de fluorescência a 50 a 400x. </li></ol> 5. P1 Neonatal Murino Ventricular Cardiomyocytes para Recellularization <ol><li> Spray de cada filhote com 70% de etanol e decapitar com uma única lâmina de uso. </li><li> Segure cada filhote entre o polegar eo indicador, enquanto o tórax é dividido na linha média com pequenas tesouras estéreis para expor a cavidade torácica. Aplicar pressão para permitir que o coração se projetam livremente do peito, enquanto ele é cortado livre dos grandes vasos e os átrios deixando apenas o tecido ventricular. </li><li> Colocar cada coração directamente para um tubo de 50 ml contendo 20 ml de cálcio arrefecido com gelo, solução salina tamponada de Hank livre de bicarbonato com 4- (2-hidroxietil) 20 mM -1-piperazinoetanossulfónico (CBFHH) até que todos os corações são recolhidos. </li><li> Aspirar o CBFHH e adicionar 10-15 ml de CBFHH fresca (gelada) para o tubo. Lavar o tecido por agitação do tecido em tele tubo várias vezes. </li><li> Decantar a solução contendo os corações em um 60 milímetros de plástico de Petri. </li><li> Dissecar qualquer tecido atrial latente dos corações sob ampliação no gelo aparando-lo longe dos corações com a tesoura primavera. Picar-se os ventrículos em pedaços pequenos tamanho homogéneo. Remover qualquer tecido não-ventricular separado do prato com # 5 fórceps. </li><li> Pipetar tanto da solução CBFHH como é necessário para transferir os fragmentos de tecido em um pequeno frasco esterilizado que contém uma barra de agitação micro estéril. </li><li> Permitir que o tecido para sedimentar para o fundo do frasco e remover a solução CBFHH. </li><li> Perfaz-se 50 ml de uma solução de enzima de 1,75 mg / ml de tripsina, 20 ug / ml de DNase II em CBFHH. Pipetar 5 ml desta solução de enzimas e adicioná-lo para um tubo contendo o tecido ventricular e picada lugar num agitador magnético. Agita-se a uma taxa constante (340 rpm) à temperatura ambiente durante 8 min. NOTA: A ação agitação deve ser suave e ainda permitirpara a suspensão da pasta. </li><li> Retirar o frasco da placa de agitação e permitir que a pasta fluida a sedimentar durante 3 min. Pipeta e descartar o sobrenadante inicial digerir, pois ele contém principalmente células do sangue e restos de tecido. </li><li> Pipetar uma segunda alíquota de 5 ml da solução de enzima e adicioná-lo ao ventrículo digestão. Agita-se durante 8 minutos e permitir que a sedimentar durante mais 3 min. Antes de iniciar as digestões, preparar dois tubos de 50 mL (rotulados 1 e 2), cada um contendo 12 ml de soro fetal de bovino gelada (FBS). Coloque um filtro de células 40 mm no topo de cada tubo. Pipet esse sobrenadante para o filtro no tubo 1. </li><li> Repetir o processo de digestão um adicional de 8 vezes, alternando-se a colocação do sobrenadante Digest entre os dois tubos contendo FBS. Dividir o último sobrenadante igualmente entre tubos 1 e 2 para garantir volumes iguais em cada tubo. NOTA: Cada tubo de recolha de agora irá conter 32,5 ml volume total de suspensão de células. </li><li> Centrifugar os tubos contêmção do sobrenadante a 150 xg durante 6 minutos a 4 ° C. </li><li> Durante a recolha das alíquotas de digestão, preparar cinco placas de cultura de tecidos de 100 mm com 6 ml de meio (de Eagle Modificado por Dulbecco meios (DMEM) com 10% FBS, 1x Pen-Strep, 1x L-glutamina) por placa. Incubar estas alíquotas a 37 ° C, 5% de CO 2 durante 30 minutos para equilibrar os meios de comunicação. </li><li> Aspirar a mistura de enzimas CBFHH e ressuspender as células em 30 ml de meio de cultura preparado acima, combinando as células de ambos os tubos de recolha. </li><li> Retorno de 6 ml de suspensão celular a cada uma das placas de 100 mm e incubar durante 45 min a 37 ° C. </li><li> No final da incubação, transferir as células não aderentes a 5 novos pratos e re-incubar durante 45 minutos (a fracção de fibroblastos terá começado a aderir aos pratos e podem ser cultivadas separadamente, se desejado). </li><li> No final da segunda rodada de pré-chapeamento, recolher os meios de comunicação e as células não aderentes de pratos e girar os meios de comunicação em 150xg durante 6 min. </li><li> Colhem-se o excesso de meios para levar as células à concentração desejada aproximada, (4,0 x 10 5 células por construto em 100 ul de perfusato). Retirar uma alíquota para contagem e ou a viabilidade de testes 14. </li></ol> 6. biorreator Recellularization de P3 coração Matrix <ol><li> Pretreat a matriz de coração isolado com meios de cultura (ver Passo 5,15) durante a noite antes de adicionar as células. </li><li> Montar os elementos de um sistema de Langendorff, como mostrado na Fig. 2. Autoclave as peças de vidro e óxido de etileno esterilizar as peças de plástico para garantir a esterilidade. Vários sub-unidades do sistema pode ser montado numa câmara de fluxo laminar antes de ser montado no cavalete de suporte. Circulating banho de água e trajecto de escoamento de água aquecida foi omitida para maior clareza. </li><li> Encha o sistema montado com 120 ml de meio de cultura de tecidos (ver passo 5.15). </li><li> Colocar a matriz de coração cateterizada do Passo 2,14 em um cul 60 milímetrostura Prato sob ampliação e ligar uma seringa de 1 mL com 22 L x 1 agulha carregada com suspensão de células a partir do Passo 5,20 para o cateter. Certifique-se de que esta montagem é livre de bolhas de ar para evitar embolizante o coração. </li><li> Suavemente perfundir os 100 ul de suspensão de células no interior da matriz do coração através das artérias coronárias. Depois de concluído, retire a combinação seringa / agulha. NOTA: A perfusão lenta de aprox. 20? L por min é necessária para impedir que as células se escoe para fora das veias, que transitam pelo coração. </li><li> Com um topo romba 22g fixada ao encaixe de Luer sobre o lado de baixo da tampa frasco de biorreactor coração, empurrar o cateter para o topo. </li><li> Iniciar a bomba peristáltica e observa-se que o fluxo procede conforme descrito na Figura 2. Rendimentos fluir a partir do reservatório através da bomba através do borbulhador para o cateter colocado na aorta no Passo 2.5 (vermelho caminho na Fig. 2). Observar o fluxo da circulação cardíaca fora da veias e gotejamento a partir do vértice, para recirculação do reservatório meios. NOTA: A taxa de fluxo de perfusão é dependente de factores individuais, tais como a resistência vascular da construção, e o tamanho do coração, mas uma taxa de 50 a 100 ul / min é um bom ponto de partida. Em pontos de tempo intermédios, avaliação funcional pode ser executado. A escala de tamanho do coração repovoados neonatal limita as avaliações, o que pode ser realizado, mas nós determinamos que os sistemas baseados opticamente pode ser utilizado para quantificar a bater comportamento do mesmo modo que foram usadas em corações de ratos adultos. 4 A construção pode ser perfundido para estendida períodos de tempo (até 23 h). </li></ol>

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The authors have nothing to disclose.

A dependência desta técnica em perfusões repetidas do coração faz a prevenção de uma embolia um componente crítico de um bom resultado. A partir do cateterismo do coração inicial nos Passos 2,2-2,6, para as mudanças de solução entre as etapas 2.8-2.14, existem manipulações que podem permitir a introdução de bolhas de ar que comprometem o fluxo de perfusato no miocárdio. Devido ao tamanho diminuto do coração neonatal, mesmo minúsculas bolhas na vasculatura pode causar um enfarte técnica, tornando as...

A insuficiência cardíaca é comum e mortal. É uma doença progressiva que resulta na diminuição da contractilidade do coração, o que prejudica o fluxo sanguíneo para os órgãos e deixa as necessidades metabólicas do corpo não satisfeitas. Estima-se que 5,7 milhões de americanos têm insuficiência cardíaca e é a principal causa de hospitalização nos Estados Unidos 9. O custo coletivo de tratamento de pacientes com insuficiência cardíaca nos Estados Unidos ultrapassa os US $ 300 bilhões de dólares por ano 9-10. O único tratamento definitivo para a insuficiência cardíaca em fase terminal é o transplante cardíaco ortotópico. Cada ano, estima-se que mais de 100.000 corações transplantados são necessários para os procedimentos de transplante cardíaco nos Estados Unidos 1-2. Devido ao número limitado de doadores, apenas cerca de 2.400 transplantes são realizados a cada ano em os EUA 2. Claramente, esta escassez de órgãos precisa ser tratada como outras estratégias são necessários para produzir órgãos adicionais para transplantação e, idealmente, estes órgãos seriam autóloga, de modo a evitar as complicações associadas com a rejeição e imunossupressão vida. Cardiomiócitos adultos mamíferos demonstrar uma capacidade de regeneração limitada após lesão, mas evidências recentes sugerem que os corações neonatais mamíferos manter uma notável capacidade de regeneração após lesão 5-8. Especificamente, após a ressecção cirúrgica parcial, uma janela de regeneração foi descoberto entre o dia do nascimento e pós-natal 7. Este período de regeneração é caracterizada por uma falta de cicatriz fibrótica, formação de neo vascularização, libertação de factores angiogénicos a partir do epicárdio, e proliferação de cardiomiócitos 8/5 , 11. Esta janela de tempo regenerativa fornece o potencial para a utilização do coração neonatal como uma nova fonte de material para o desenvolvimento de um coração bioartificial. A matriz extracelular é conhecida por fornecer pistas importantes para promover cardiomyocyte a proliferação e crescimento. Diferenças distintas na disponibilidade de moléculas nas matrizes neonatais e adultos de 12 e sua capacidade para promover a regeneração foram exploradas 13. matrizes adultos descelularizados têm sido utilizados em vários estudos para fornecer um andaime ECM para repovoamento celular e a geração de um coração bioartificial. Embora esses estudos, e novas descobertas em tecnologias de células-tronco, estão avançando rapidamente, vários obstáculos ainda precisam ser cumpridos. Por exemplo, as limitações em preservando a estrutura nativa da matriz, a integração celular na parede da matriz, e capacidade para suportar a proliferação e crescimento de todos os limites do sucesso desta abordagem. Enquanto atributos regenerativos superiores têm sido atribuídas ao coração neonatal, os aspectos práticos da utilização de um tecido tal têm limitado a sua exploração. Com base na capacidade regenerativa demonstrada do coração neonatal, desenvolvemos novas matrizes através do desenvolvimento de umtécnica de descelularização para o coração do rato P3. O coração P3 foi escolhido para estes estudos, uma vez que está dentro da janela de regeneração cardíaca como anteriormente determinada 6 mas o coração é suficientemente grande para colheita, decellularize e recellularize. O objetivo deste estudo é demonstrar a viabilidade da criação de uma matriz de um coração neonatal mouse. Os nossos estudos fornecem evidência para a viabilidade de um minuto descelularizante, coração neonatal, mantendo a integridade estrutural e proteinácea do ECM. Nós também demonstrar a capacidade de recellularize este ECM cardíaca com mCherry cardiomiócitos expressam e examinamos esses cardiomiócitos para a expressão de vários marcadores cardíacos seguintes recelularização. Esta tecnologia permitirá que para o ensaio da superioridade de uma matriz neonatal para o desenvolvimento de um coração bioartificial.

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. Materials for mouse heart isolation&lt;/strong&gt;</td></tr><tr><td>P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J)</td><td>Jackson Laboratories</td><td>21577</td><td>or equivalent</td></tr><tr><td>60 mm Culture dish</td><td>BD Falcon</td><td>353004</td><td>or equivalent</td></tr><tr><td>Phosphate buffered saline pH 7.4 (sterile)</td><td>Hyclone</td><td>SH30256.01</td><td>or equivalent</td></tr><tr><td>Single Use Blade</td><td>Stanley</td><td>28-510</td><td>or equivalent</td></tr><tr><td>Standard Scissors</td><td>Moria Bonn (Fine Science Tools)</td><td>14381-43</td><td>or equivalent</td></tr><tr><td>Spring Scissors 10 cm</td><td>Fine Science Tools</td><td>15024-10</td><td>or equivalent</td></tr><tr><td>Vannas Spring Scissors - 3 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-00</td><td>or equivalent</td></tr><tr><td>#5 Forceps</td><td>Dumnot (Fine Science Tools)</td><td>11295-00</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;2. Materials for decellularization&lt;/strong&gt;</td></tr><tr><td>Inlet adaptor</td><td>Chemglass</td><td>CG-1013</td><td>autoclavable</td></tr><tr><td>Septum</td><td>Chemglass</td><td>CG-3022-99</td><td>autoclavable</td></tr><tr><td>1/8 in. ID x 3/8 OD C-Flex tubing</td><td>Cole-Parmer</td><td>EW-06422-10</td><td>autoclavable</td></tr><tr><td>Male luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K33</td><td>autoclavable</td></tr><tr><td>Female luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K26</td><td>autoclavable</td></tr><tr><td>Prolene 7-0 surgical suture&amp;nbsp;</td><td>Ethicon</td><td>8648G</td><td>or equivalent</td></tr><tr><td>Ring stand</td><td>Fisher Scientific</td><td>S47807</td><td>or equivalent</td></tr><tr><td>Clamp</td><td>Fisher Scientific</td><td>05-769-6Q</td><td>or equivalent</td></tr><tr><td>Clamp regular holder</td><td>Fisher Scientific</td><td>05-754Q</td><td>or equivalent</td></tr><tr><td>60 cc syringe barrel&amp;nbsp;</td><td>Coviden</td><td>1186000777T</td><td>or equivalent</td></tr><tr><td>Beaker</td><td>Kimble</td><td>14000250</td><td>or equivalent</td></tr><tr><td>22 G x 1 Syringe Needle</td><td>BD</td><td>305155</td><td>or equivalent</td></tr><tr><td>12 cc syringe</td><td>Coviden</td><td>8881512878</td><td>or equivalent</td></tr><tr><td>3-way stop cock</td><td>Smith Medical</td><td>MX5311L</td><td>or equivalent</td></tr><tr><td>PE50 tubing</td><td>BD Clay Adams Intramedic</td><td>427411</td><td>Must be formable by heat. Polyethylene recommended.</td></tr><tr><td>1% SDS</td><td>Invitrogen</td><td>15525-017</td><td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.</td></tr><tr><td>1% Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787</td><td>Make up fresh solution from a 10% stock and filter sterilize before use.&amp;nbsp;</td></tr><tr><td>Sterile dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Hyclone</td><td>SH30538.02</td><td>Or MilliQ system purified water.</td></tr><tr><td>1x Pen/Strep</td><td>Corning CellGro</td><td>30-001-Cl</td><td>or equivalent&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;3. Materials for DNA quantitation&lt;/strong&gt;</td></tr><tr><td>Proteinase K</td><td>Fisher</td><td>BP1700</td><td>&gt;30 U/mg activity</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>or equivalent</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Mallinckrodt</td><td>5958-04</td><td>or equivalent</td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1379</td><td>or equivalent</td></tr><tr><td>Tris base/hydrochloride</td><td>Sigma-Aldrich</td><td>T1503/T5941</td><td>or equivalent</td></tr><tr><td>Pico-Green dsDNA assay kit</td><td>Life Technologies&amp;nbsp;</td><td>P7589</td><td>requires fluorimeter to read</td></tr><tr><td colspan="4">&lt;strong&gt;4. Method for fixation and sectioning of tissue&lt;/strong&gt;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>or equivalent</td></tr><tr><td>Gelatin Type A from porcine skin</td><td>Sigma-Aldrich</td><td>G2500</td><td>must be 300 bloom or greater</td></tr><tr><td colspan="4">&lt;strong&gt;5. Method for tissue histology&lt;/strong&gt;</td></tr><tr><td>Cryomolds 10 x 10 x 5 mm</td><td>Tissue-Tek</td><td>4565</td><td>or equivalent</td></tr><tr><td>Cryostat</td><td>Hacker/Bright</td><td>Model OTF</td><td>or equivalent</td></tr><tr><td>Microscope Slides&amp;nbsp; 25 x 75 x 1 mm</td><td>Fisher Scientific</td><td>12-550-19</td><td>or equivalent</td></tr><tr><td>Hematoxylin 560&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801570</td><td>or equivalent</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2701</td><td>or equivalent</td></tr><tr><td>Define</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3803590</td><td>or equivalent</td></tr><tr><td>Blue buffer&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3802915</td><td>or equivalent</td></tr><tr><td>Alcoholic Eosin Y 515&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801615</td><td>or equivalent</td></tr><tr><td>Formula 83 Xylene substitute&amp;nbsp;</td><td>CBG Biotech&amp;nbsp;</td><td>CH0104B</td><td>or equivalent</td></tr><tr><td>Permount Mounting Medium&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>SP15-500</td><td>or equivalent</td></tr><tr><td>Collagen IV Antibody</td><td>Rockland</td><td>600-401-106.1</td><td>or equivalent</td></tr><tr><td>&amp;alpha;-Actinin Antibody</td><td>Abcam</td><td>AB9465</td><td>or equivalent</td></tr><tr><td>mCherry Antibody</td><td>Abcam</td><td>AB205402</td><td>or equivalent</td></tr><tr><td>NKX2.5 Antibody</td><td>Santa Cruz Biotechnology</td><td>SC-8697</td><td>or equivalent</td></tr><tr><td>Donkey anti-mouse AF488 Antibody</td><td>Life Technology</td><td>A21202</td><td>or equivalent</td></tr><tr><td>Donkey anti-chicken AF594 Antibody</td><td>Jackson Immunoresearch</td><td>703-585-155&amp;nbsp;</td><td>or equivalent</td></tr><tr><td>Donkey anti-goat CY5 Antibody</td><td>Jackson Immunoresearch&amp;nbsp;</td><td>705-175-147</td><td>or equivalent</td></tr><tr><td>Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td><td>Jackson Immunoresearch</td><td>111-587-003</td><td>or equivalent</td></tr><tr><td>Prolong Gold Antifade Mountant with DAPI</td><td>ThermoFisher</td><td>P36930</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;6. Isolation of neonatal ventricular cardiomyocytes using pre-plating&lt;/strong&gt;</td></tr><tr><td>HBSS (Ca, Mg Free)</td><td>Hyclone</td><td>SH30031.02</td><td>or equivalent</td></tr><tr><td>HEPES (1 M)</td><td>Corning CellGro</td><td>25-060-Cl</td><td>or equivalent</td></tr><tr><td>Cell Strainer</td><td>BD Falcon</td><td>352340</td><td>or equivalent</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>or equivalent</td></tr><tr><td>Primeria 100 mm plates</td><td>Corning</td><td>353803</td><td>Primeria surface enhances fibroblast attachment promoting a higher myocyte purity</td></tr><tr><td>Trypsin</td><td>Difco</td><td>215240</td><td>or equivalent</td></tr><tr><td>DNase II</td><td>Sigma-Aldrich</td><td>D8764</td><td>or equivalent</td></tr><tr><td>DMEM (Delbecco&#039;s Minimal Essential Media)</td><td>Hyclone</td><td>SH30022.01</td><td>or equivalent</td></tr><tr><td>Vitamin B12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>V6629</td><td>or equivalent</td></tr><tr><td>Fibronectin coated plates&amp;nbsp;</td><td>BD Bioscience</td><td>354501</td><td>or equivalent</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Hyclone</td><td>SH30910.03</td><td>or equivalent</td></tr><tr><td>Heart bioreactor glassware</td><td>Radnoti Glass Technology</td><td>120101BEZ</td><td>Must be sterilizable by autoclaving or gas.</td></tr></tbody></table>

neonatal cardiac scaffolds: novel matrices for regenerative studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

decelularização Em média, o tempo para a descelularização de um coração P3 utilizando este protocolo é de aproximadamente 14 horas. dado um peso médio do coração de 23 mg para o recém-nascido P3. acelularidade Figura 3a mostra um coração neonatal P3 totalmente intacto (conjunto de montagem). Figura 3b mostra o mesmo coração seguinte descelulariza?...

Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Nestes estudos, que fornecem metodologia para novos, neonatal, andaimes cardíacas murinas para uso em estudos de regeneração.

Neonatal Cardiac Scaffolds: Matrizes novas para Estudos Regenerativa

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

neonatal-cardiac-scaffolds-novel-matrices-for-regenerative-studies

Research

JoVE Journal

Bioengineering

7.7K visualizações.  University of Minnesota.  Nestes estudos, que fornecem metodologia para novos, neonatal, andaimes cardíacas murinas para uso em estudos de regeneração. 

Vídeo: Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

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 ...

Bioengenharia

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Engenharia de Tecidos: Construção de um multicelular 3D andaime para a entrega de camadas celulares Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Fabricação de Miogênica Constrói engenharia de tecidos

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Biologia

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Decellularization comparável de explantes de tecido cardíaco Fetal e adulto como plataformas 3D-como para estudos de Vitro

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Um método baseado em molde líquido de criação livre de andaime tridimensional de tecido cardíaco

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabricação de matriz de cartilagem-derivado Decellularized andaimes

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Preparação de tecidos cardíacos projetados em forma de malha derivados de células iPS humanas para reparo de miocárdio in vivo

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Geração, Triagem de Alto Rendimento e Biobanco de Esferoides Cardíacos Derivados de Células-Tronco Pluripotentes Induzidos por Humanos.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

Geração de tecido miocárdico humano 3D usando escrita eletrogiratória por fusão de andaimes de policaprolactona e células cardíacas derivadas de hiPSC

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

Transplante de coração in vivo para estudar a transição endotelial para mesenquimal

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Autor em destaque: Um modelo de transplante cardíaco de rato heterotópico neonatal para o estudo da transição endotelial para mesenquimal  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Modelo de Transplante Cardíaco Heterotópico Neonatal em Rato para Estudo da Transição Endotelial-Mesenquimal

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...