Os autores agradecem ao Prof Hendrik Tevaearai e do Departamento de Cirurgia Cardiovascular de apoio financeiro para a parte inicial do estudo.

NOTA: Lewis ratos machos e fêmeas, de 200-220 g foram alojados em condições padrão de laboratório (12 horas de luz e de ciclo de escuro, de água ad libitum e alimentares, IVC gaiola). Todos os animais foram tratados de acordo com as recomendações do FELASA ea Lei Suíça sobre a protecção animal. 1. celular Preparação: Isolamento de Células-Tronco Mesenquimais de Medula Óssea <ol><li> Anestesiar os ratos com isoflurano a 5% de O 2 e 5 L / min, numa câmara de indução por 5 min. Coloque focinho do animal, para um cone de nariz ligada ao sistema de anestesia. Realize um dedo do pé ou cauda pitada de confirmar sedação. </li><li> Retirar a pele da perna com uma tesoura de corte da pele do tornozelo para o quadril. Retirar os músculos e corte da artéria femoral para a exsanguinação animal. Expor a articulação do quadril e deslocar a cabeça do fêmur. É importante para não danificar a cabeça do fêmur. Colocar os ossos em PBS estéril num tubo de plástico de 50 ml. </li><li> Remova todas as muscles e ligamentos dos ossos sem quebrá-los. NOTA: É importante que o osso permanece intacto para evitar ligação de solução de lavagem ou de contaminação com etanol. Lave os ossos pelados em etanol 70% por 5 min. </li><li> Sob a estéril câmara de fluxo laminar, cortar ambas as pontas dos ossos com uma tesoura. Lave a medula óssea através da injeção de PBS estéril a partir da extremidade do osso. Recolha de medula para um tubo de 15 ml. Centrífuga 7 min a 300 x g. </li><li> Remover o sobrenadante. Suspender o sedimento em 3 ml de tampão de lise de glóbulos vermelhos. Incubar a suspensão durante 1 min à temperatura ambiente antes de girar de novo durante 7 minutos a 300 x g. Em um frasco de cultura de 150 ml, estéril adicionar meio de cultura celular (395 ml de meio IMDM, 5 ml de pen / strep, 100 ml de FBS) e propagar as células. </li><li> Alterar o segundo meio dia para remover células não aderentes. <ol><li> Realizar as mudanças de meio a cada segundo dia, durante 2-3 semanas, até que a quantidade de célula desejado é obtido. </li><li> Em alternativa, preparar o biograft por sementesção das células para a matriz sólida, como descrito noutro local necessário e 3. </li></ol></li><li> O dia da implantação cardíaca, preparar as células ou colher o biograft imediatamente antes da aplicação do epicárdio. <ol><li> Coletar as células usando solução desprendimento de células Accutase. Contar as células. Encher um tubo de centrífuga de 1,5 ml, com o volume de suspensão de células para obter uma quantidade calculada de célula desejado. Centrifuga-se durante 7 minutos a 300 xg e remover o sobrenadante. </li><li> Alternativamente, a colheita biograft de meio de cultura celular, enxaguar em PBS estéril, e manter em meio de cultura fresco sem soro. </li></ol></li></ol> 2. Primeiro toracotomia e LAD Ligadura <ol><li> Pesa-se o rato. Ligue o bloco de aquecimento a 37 ° C. Anestesiar ratos com 5% de isoflurano e 5 L / min de 100% de O 2 em uma câmara de indução para 5-7 min. Realize um dedo do pé ou cauda pitada de confirmar sedação. Coloque o rato sobre a almofada de aquecimento. </li><li> Entubar o animal com um 14L iv cateter. Conectar o cateter intubação a um ventilador roedor programado para 2,5 L / min de oxigênio, 2,5% de isoflurano, o volume corrente de 2 ml, e uma freqüência respiratória de 90 ciclos / min. </li><li> Prepara-se uma solução de buprenorfina a 0,1 mg / kg. Injectar subcutaneamente um terço da solução. Raspar a parte esquerda do tórax. Desinfectar com uma solução de Betadine 1%. </li><li> Incisar a pele perpendicular ao esterno no quarto espaço intercostal. Separe as três camadas de músculo torácico (peitoral maior, peitoral ascendente e peitoral oblíquo externo). Abra o quarto espaço intercostal (entre as costelas 4 e 5). </li><li> Use um pequeno afastador para espalhar as costelas e para expor o coração. Abra o pericárdio com cuidado. Localize e ligadura da artéria descendente anterior coronariana (LAD), com 7,0 sutura 4 milímetros abaixo do átrio. </li><li> Feche o espaço intercostal, com dois pontos com 3,0 suturas. Posicione as duas suturas proximal e distal do esterno. Em primeiro lugar, tighten sutura distal. Prenda o tubo de escape ventilador para 2 segundos, a fim de inflar o peito e evitar pneumotórax. Aperte a segunda sutura. Posicione as camadas de músculos de volta no lugar (sem suturas são necessários). Fechar a pele com uma sutura 5.0. </li><li> Desinfetar a sutura com solução Betadine 1%. Injectar o resto da solução de buprenorfina. Desligue o sistema de anestesia. Remover cateter intubação. </li><li> Mantenha o rato na gaiola sob uma lâmpada quente por 1-2 horas. Manter um termómetro na gaiola e controlar a distância entre a lâmpada e o compartimento quente, a fim de evitar um excesso de aquecimento. Retorne o rato na unidade IVC. Injetar por via subcutânea Buprenorfina 0,05-0,1 mg / kg pós -operatively cada 6-12 horas para 48 horas. </li></ol> 3. Epicárdica Administração da Via Tratamento Segunda toracotomia <ol><li> Repita os passos de 2,1-2,3. </li><li> Incisar a pele perpendicular ao esterno no quinto espaço intercostal. Separar a fase 3s do músculo torácico (peitoral maior, peitoral ascendente e peitoral oblíquo externo). Abra o quinto espaço intercostal (entre as costelas 5 e 6). </li><li> Use um pequeno afastador para espalhar as costelas e para expor o coração. Se há alguma adesão, retire-os cuidadosamente com uma pinça fina. </li><li> Localize a área do infarto que aparece como uma área clara abaixo da ligadura. NOTA: Se necessário, use um pedaço de 10 cm de sutura (7,0) inserido no ápice para melhor visualizar o ventrículo esquerdo. Puxe delicadamente à sutura mantida frouxamente por uma braçadeira para expor o coração. </li><li> Aplique um dos seguintes tratamentos: <ol><li> Posicionar o biograft na superfície do coração. Utilizar a seringa pré-cheia de selante de fibrina previamente aquecida à temperatura ambiente durante 10 min. Aplique uma gota (50-100 L) de selante de fibrina sob a biograft usando uma agulha 25 G Luer. Verifique se o biograft está perfeitamente selado. </li><li> Alternativamente, aplicam-se o sedimento de células recolhidas com uma pipeta de ponta na superfície of o coração na área abaixo da ligadura pálido. Aplicar uma gota (50-100 ul) de selante de fibrina sobre o sedimento celular. </li></ol></li><li> Remover o pedaço de fio de sutura a partir do vértice. </li><li> Repita os passos de 2,6-2,8. </li></ol>

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The authors have nothing to disclose.

A ligadura permanente da LAD causa lesão miocárdica irreversível. O primeiro modelo animal foi descrito em 1960 4. Desde então, tem sido considerado como um modelo padrão e adequado para MI crónica. Sua estabilidade e reprodutibilidade permitiu a avaliação experimental de terapias para MI 5. Procedimentos melhorou após a descrição inicial relatou uma taxa de mortalidade operatória de 35-13% 6. Como esperado, a ligadura LAD induzida função cardí...

Desde o final do século 19, a doença cardiovascular continua a ser o número um assassino em série nos países industrializados. Entre eles, a doença arterial coronária representa a etiologia principal. Os resultados de fase aguda no infarto do miocárdio (MI) e é seguido por remodelação mal-adaptativo que se desenvolve progressivamente em direção a uma fase crônica e insuficiência cardíaca grave. Apesar dos recentes avanços tecnológicos e terapêuticos significativos, a morbidade ea mortalidade devido à progressão da insuficiência cardíaca ainda está crescendo 1. Neste contexto, a terapia celular tem ganhado cada vez mais interesse como uma nova opção terapêutica para parar a progressão da doença para a insuficiência cardíaca e para estimular a capacidade regenerativa recentemente identificado do miocárdio. Estudos experimentais e clínicos têm fornecido muitas evidências dos efeitos benéficos obtidos após o transplante cardíaco de vários tipos de células. Entre os principais resultados incluíram melhoria cardifunção contrátil ac, diminuição do remodelamento ventricular esquerdo, redução do tamanho do infarto, e aumento da densidade vascular na área infartada. No entanto, a baixa retenção número de células após a injeção de células manteve-se uma desvantagem importante. Associação de células com um biomatriz para melhorar de entrega célula 2, recentemente promovido pesquisador e interesses clínicos. Ligadura da artéria descendente anterior da artéria coronária (LAD) é um método de referência para MI em modelo animal de pequeno porte que resulta em infarto transmural e uma cicatriz madura. A terapia celular aplicada na fase crônica da MI requer uma segunda intervenção cirúrgica. A esternotomia mediana geralmente é realizada para permitir a injeção intramiocárdica das células ou a implantação do epicárdio de bioenxertos. Tais procedimentos cirúrgicos invasivos, aumentar a taxa de mortalidade, pós-cirurgia de tempo de recuperação, a dor, e o risco de infecção. A abordagem minimamente invasiva aqui apresentada não só evita esse viés, mas também fornece oacessibilidade ptimal do coração para a aplicação dos tratamentos. MI e implantação do epicárdio de células associadas com uma biomatriz tipo gel são realizadas com o coração batendo através de toracotomia intercostal esquerdo.

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			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
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			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
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			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
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			<td>TISSEEL Glue, Frozen -20&#176;C</td>
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			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
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			<td>use as retractor for the ribs</td>
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			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
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			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
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			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
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			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
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			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
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			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
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cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

Terapia celular cardíaca tem ganhado interesse crescente e implante de biomateriais associados com as células se tornou uma questão importante para otimizar a entrega de células do miocárdio. Roedor modelo de infarto do miocárdio (IM), que consiste de Esquerda artéria descendente anterior (LAD) ligadura tem sido comumente realizada através de uma toracotomia; uma segunda cirurgia de coração aberto através de uma esternotomia tem sido tradicionalmente realizada para a aplicação do epicárdio do tratamento. Desde a descrição do modelo de ligadura LAD, a taxa de mortalidade pós-operatória diminuiu 35-13%, porém a segunda cirurgia manteve-se crítico. A fim de melhorar a recuperação pós-cirurgia e reduzir a dor e infecção, procedimentos cirúrgicos minimamente invasivos são apresentados. Dois toracotomia foram realizadas, a inicial para LAD ligadura e o segundo tratamento a administração de epicárdico. Bioenxertos consistindo de células associadas com matrizes de tipo sólido ou de gel foram aplicados na AR enfartadaea. LAD ligação resultou em perda da função cardíaca, como confirmado por ecocardiografia realizada após 2 e 6 semanas. Goldner tricrômico coloração realizada em secções coração confirmou a formação de cicatriz transmural. Cirurgias primeiro e segundo resultou em menos que 10% de mortalidade pós-operatória.

Todos os animais recuperados dentro de 1 hora após toracotomias. A cicatrização foi rápida. Não se observou qualquer edema ou infecção. A toracotomia esquerda dobro permitido o acesso ideal para o coração (Figura 1). Dor e mortalidade pós-operatória foi baixa. O animal recuperou-se rapidamente a partir da cirurgia e ganho de peso (Figura 2). A percentagem de sobrevivência de Kaplan Meir foi de 96% para o primeiro toracotomia. Quatro ratos mais de...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Implantação de um biograft para tratar infarto do miocárdio induzido por ligadura LAD em um modelo de roedor tem convencionalmente necessárias duas cirurgias de coração aberto. A fim de reduzir a mortalidade e proporcionar as condições óptimas para a fixação de Biomatrizes sólidos gelatinosos e associados a células, procedimentos minimamente invasivos têm sido desenvolvidos.

Terapia Celular para insuficiência cardíaca em Rat: Double toracotomia para infarto do miocárdio e Epicárdica implante de células e Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K visualizações.  University of Fribourg.  Implantação de um biograft para tratar infarto do miocárdio induzido por ligadura LAD em um modelo de roedor tem convencionalmente necessárias duas cirurgias de coração aberto. A fim de reduzir a mortalidade e proporcionar as condições óptimas para a fixação de Biomatrizes sólidos gelatinosos e associados a células, procedimentos minimamente invasivos têm sido desenvolvidos. 

Vídeo: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imagem bioluminescência para avaliação das respostas imune após o implante do tecido do coração Engineered (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Bioengenharia

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplante de Progenitores Miogênicos derivados de células-tronco pluripotentes induzidos em modelos de regeneração muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Análise da Membrana Tubular Networks em Cardiac Miócitos de átrios e ventrículos

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

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

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

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

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

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

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Avaliação da proliferação queratinócito em duas e três dimensões tipo I colágeno substratos

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Microbolhas magnéticas, acústicas e óptica-tripla-responsivas para hipertermia magnética e terapia de câncer de combinação pothototérmica

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

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 ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplante de um Patch Bioimpresso 3D em um modelo murino de infarto do miocárdio

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Quantificação da função ventricular esquerda do coração do rato, tensão do miocárdio e forças hemodinâmicas por ressonância magnética cardiovascular

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Avaliação da Terapia de Modulação da Contratilidade Cardíaca em Cardiomiócitos Derivados de Células-Tronco Humanas 2D

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...