Os autores querem agradecer Yabin Wu para cultura dos tecidos apresentados na Figura 2, a foto foi tirada por Bart van Overbeeke. O trabalho foi financiado pela SenterNovem, ISO 42022 concessão.

1. Cultura de Células de mioblastos de murino progenitoras ou células C2C12 <ol><li> Isolar as células de acordo com o protocolo inicialmente publicado por Shefer e colegas 10 e posterior adaptada por Collins et al. 11 e Boonen et al. 12 e armazená-los em azoto líquido. Isto requer ratos, por exemplo, C57BL / 6. Os métodos alternativos são usadas em outros laboratórios, por exemplo, um método publicado em Journal of Experiments visualizadas por Li Y et al. 13. Para os reagentes e equipamentos que são referidos na tabela da página 7 e 8. A partir do músculo de um rato irá geralmente obter células suficientes para criopreservar cerca de 20 frascos em azoto líquido. Em seguida, inicia o protocolo de descongelar as células a partir da armazenagem de azoto líquido. </li><li> Colocar as células a partir de um frasco para um 25 cm 2 Matrigel (1 mg / ml) frasco de cultura de tecido revestido e adicionar meio de crescimento (GM) que consiste em (335 ml de DMEM avançada, 100 ml fetal bovine soro (FBS), 50 ml HS, 5 ml penicilina / estreptomicina, 5 ml de L-glutamina, 5 ml de extracto de embriões de galinha (CEE)). </li></ol> Nota: revestimento durante 2 h à temperatura ambiente e remover o Matrigel por aspiração. <ol start="3"><li> Subcultura das células de aproximadamente 1:03 a cada 3 dias. Isso significa que: no dia 3: células passagem de um 25 centímetros 2 para um frasco de 75 cm 2, no dia 6: as células de passagem de um frasco de 75 cm 2 para dois 150 centímetros 2 frascos (com 30 min preplating em frascos sem revestimento). No dia 9 de transferência de um balão de 150 centímetros 2 a 1 150 centímetros triplo Frasco de 2, ou se não estiver disponível a três frascos de 150 centímetros 2 (se necessário preplate novamente dependendo do fenótipo das células). No dia 12, as células estão prontas para semear em uma construção muscular. </li></ol> Notas: - Por triplo 150 cm 2, o número de células será de aproximadamente 4,5 x 10 6 células. - Use vials de isolamentos diferentes para misturá-las para se obter uma população mista de células na altura da sementeira. Nota: Se você optar por não trabalhar com células primárias, mioblastos C2C12 são uma boa alternativa. 2. Engenharia de tecidos músculo esquelético <ol><li> Prepare cola de silicone misturando-se o &quot;elastómero&quot; com o &quot;agente de cura&quot; (10:1). Cortar + / - 5 mm segmentos quadrados de velcro com um lado triangular (casa-como a forma, a Figura 1). Cola o velcro para os poços de uma placa de cultura de 6 poços em um espaço de aproximadamente 12 mm entre os quadrados. </li></ol> Notas: - Só use o lado macio do velcro e enfrentar esta para cima o lado. - Certifique-se de que os telhados se enfrentam. - Apenas cobrir o velcro com cola de silicone, não espalhar cola por todo o bem. - Para a estimulação elétrica depois é relevante para alinhar as construções na direção vertical do pl bemcomeu (ao longo do eixo). <ol start="2"><li> Deixa-se secar durante a noite num forno de vácuo, não aquecida, principalmente para remover as bolhas de ar. Esterilizar por adição de EtOH a 70% aos poços e incubar durante 15 min. Enxaguar 3x com PBS e colocadas sob UV, durante 15 minutos. Remover todo o PBS a partir dos poços e o Velcro e coloque na incubadora até à sua utilização. </li><li> Descongelar Matrigel solução no frigorífico e preparar a solução de colagénio para a concentração desejada, pouco antes de fazer as construções em 3D. Dilui-se o colagénio de estoque com ácido acético a 0,02% estéril (concentração final de 3,2 mg / ml). </li></ol> Nota: deixar tudo no gelo. <ol start="4"><li> Tripsinizar as células, ressuspender em GM e contagem. Deixar as células num tubo de centrifugadora na incubadora. </li><li> Adicionar a quantidade desejada de solução de colagénio para o número de construções que pretende fazer a um tubo (de acordo com a Tabela 1), adicionar NaOH 0,5 M a esta solução de colagénio até uma cor de rosa claro color é o que indica um valor de pH de 7,5. Misture suavemente pipetando para cima e para baixo e evitar a formação de bolhas. Em seguida, adicione o Matrigel e misture muito delicadamente mas completamente. Por fim, adicionar o GM-se à mistura de colagénio / Matrigel (para as quantidades apropriadas ver Tabela 1). </li></ol> Notas: - Execute cada passo no gelo! Matrigel e colagénio será prontamente gel à temperatura crescente. - Lembre-se que a cor realmente é rosa! Um aumento no pH também induzir a gelificação rápida. - A concentração final de colagénio: 1,6 mg / ml. <ol start="6"><li> Centrifugar a quantidade desejada de células a 1.000 rpm durante 5 minutos e remove-se o sobrenadante. </li></ol> Nota: - Consoante a actividade das células do número de células por construção necessita de ser ajustada. Tipicamente, o número de células situa-se entre 750.000 e 1.250.000 células por construção. <ol start="7"><li> Utilizar parte da mistura de gel para ressuspender o sedimento celulare transferir as células para a mistura restante e misturar cuidadosamente, mas sem a introdução de bolhas de ar. </li><li> Levar as placas pré-aquecidos bem fora da incubadora e pipeta 0,35 -0,4 ml da mistura de células-gel na primeira Velcro. Então, começar a pipetar a mistura a partir do centro, entre os locais de fixação e estender-se ao Velcro. Finalmente, a utilização do restante do gel para encher o espaço entre as duas escoras de velcro e de pipeta em torno dos pedaços de velcro. </li><li> Verifique cuidadosamente após 5-10 min se os géis são sólidos o suficiente para ser transferido, ou seja, bata suavemente os pratos e inspecionar visualmente se o gel é rígida. Se assim for, coloque cuidadosamente os pratos em uma incubadora. Geralmente, eles podem ser recolhidos depois de dez minutos. Em seguida, após 1-2 horas, gentilmente sobrepor cada gel com 4 mL de GM quente. </li></ol> Nota:-Não faça movimentos vigorosos ao manusear as placas. <ol start="10"><li> Substitua GM por meio de diferenciação (meio de crescimentosem extrato de embrião de galinha), após 24 horas, e substituir por meio fresco a cada 2-3 dias. No dia 7 deverá ter obtido maduros fibras musculares orientadas, como pode ser avaliado com o desenvolvimento da coloração por estrias cruzadas nas fibras musculares fundidos. </li></ol> 3. Estimulação Elétrica <ol><li> Esterilizar os eléctrodos a partir da placa ionoptix (ver tabela de reagentes e equipamento) antes da utilização, com etanol a 70% e, subsequentemente, seco sob UV numa câmara de segurança. Colocar a placa com os eléctrodos para a placa de cultura com as construções e cobrir com a tampa da placa de cultura e transferência para a incubadora. Conecte o Ionoptix C-pacer com os cabos adequados. </li></ol> Nota: Os eléctrodos são colocados em paralelo ao lado da construção muscular (Figura 2). <ol start="2"><li> Aplicar o protocolo de estimulação, como previsto. </li></ol> Nota: Nós usamos geralmente 4 V / cm,Pulsos 6ms com uma frequência de 2Hz. Alterar o meio de cultura a cada 24 h durante a estimulação.

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A engenharia de tecidos musculares tem um grande potencial para a utilização como um modelo de doença, para a triagem de drogas, em medicina regenerativa e para a produção de carne. No entanto, os requisitos para estas aplicações variar. Optou-se por trabalhar com uma combinação de colagénio e de matrigel, porque permite o alinhamento de colagénio e células porque as células progenitoras mioblastos requerem a presença de proteínas de membrana basal derivadas, tal como determinado em estudos anteriores 2D ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Matrigel factor de crescimento reduzido </td><td> Beckton Dickinson e </td><td></td><td></td></tr><tr><td> DMEM (glucose elevada) * </td><td> Gibco </td><td> 42430 </td><td></td></tr><tr><td> Avançado DMEM </td><td> Gibco </td><td> 12491 </td><td></td></tr><tr><td> Soro de cavalo </td><td> Gibco </td><td> 65050-122 </td><td></td></tr><tr><td> De soro fetal bovino </td><td> Greiner </td><td> 758075 </td><td></td></tr><tr><td> 0,45 e 0,22 um filtro de seringa * </td><td> Whatmann (Schleicher e Scheull) </td><td> 10462100 </td><td></td></tr><tr><td> L-glutamina </td><td> Gibco </td><td> 25030024 </td><td></td></tr><tr&gt; <td> Penicilina / estreptomicina </td><td> Gibco </td><td> 10378016 </td><td></td></tr><tr><td> Anfotericina </td><td> Gibco </td><td> 15290-018 </td><td></td></tr><tr><td> Plástico para cultura </td><td> Greiner </td><td></td><td> Inclui frascos de cultura e pipetas </td></tr><tr><td> Extracto de embrião de galinha </td><td> United States Biological </td><td> C3999 </td><td></td></tr><tr><td> Pipeta de Pasteur * </td><td> Hilgenberg </td><td></td><td> Pipetas Pasteur, com a constrição, com algodão L ponta, aberto: 230 mm de diâmetro com ponta de 0,9 - 1,1 mm </td></tr><tr><td> Pipeta de Pasteur * </td><td> Hilgenberg </td><td></td><td> Pipetas Pasteur, com a constrição, com algodão L ponta, aberto: 230 mm de diâmetro com ponta de 1,4 - 1,6 mm </td></tr><tr><td> Pipeta de Pasteur </td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> Colagenase tipo I* </td><td> Sigma </td><td> C0130-16 </td><td></td></tr><tr><td> 40 mM filtro célula * </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> Agulha de 19G </td><td></td><td></td><td></td></tr><tr><td> Elastômero </td><td> Dow Corning Corporation </td><td> 3097358-1004 </td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Agente de cura </td><td> Dow Corning Corporation </td><td></td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Velcro </td><td> Loja regular </td><td></td><td> Você pode comprar este em uma loja regular, usar apenas o lado macio </td></tr><tr><td> Colágeno tipo I, cauda de rato </td><td> BD Biosciences </td><td> 3544236 </td><td></td></tr><tr><td> C-Pace Pacer Cultura PE </td><td> Ionoptix </td><td></td><td></td></tr><tr><td> 6 poços de placas de cultura de estimulação eléctrica </td><td> Beckton Dickinson-Falcon </td><td> BD Falcon # 353846 </td><td></td></tr><tr><td> C-prato prato eletrodos cultura </td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> * Necessária para o isolamento de células (ponto 1.1) # Juntos em um kit </td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

Tecidos musculares engenharia pode ser utilizado para diversos fins, que incluem a produção de tecidos para utilização como um modelo de doença in vitro, por exemplo, para estudar as úlceras de pressão, para a medicina regenerativa e como uma alternativa a carne 1. As primeiras construções relatados musculares 3D foram feitos há muitos anos e pioneiros no campo são Vandenburgh e colegas 2,3. Avanços na engenharia de tecidos músculo não são apenas o resultado do ganho enorme no conhecimento de fatores bioquímicos, células-tronco e células progenitoras, mas estão em base particular em conhecimentos obtidos por pesquisadores que os fatores físicos desempenham papéis essenciais no controle do comportamento de células e tecidos de desenvolvimento. Estado-da-arte da engenharia muscular constrói atualmente consistem de construções de células-povoadas de hidrogel. No nosso laboratório que geralmente são constituídos por células mioblastos de murino progenitoras isoladas, a partir de murinos músculos dos membros posteriores ou de uma linha celular C2C12 de mioblastos de murino, mixado com uma mistura de colagénio / Matrigel e semeadas entre dois pontos de ancoragem, que imita os ligamentos musculares. Outras células podem ser considerados, bem como, por exemplo, linhas celulares alternativos, tais como os mioblastos L6 de rato neonatal musculares 4, células progenitoras, células derivadas 5 derivadas de tecidos musculares adultas a partir de outras espécies, tais como 6 humana ou células pluripotentes estaminais ainda induzidas (células iPS) 7 . Contractilidade celular faz com que o alinhamento das células ao longo do eixo longitudinal da construção de 8,9 e diferenciação das células progenitoras musculares após cerca de uma semana de cultura. Além disso, a aplicação de estimulação eléctrica pode melhorar o processo de diferenciação, em certa medida 8. Devido ao seu tamanho limitado (8 x 2 x 0,5 mm) do tecido completo pode ser analisada utilizando microscopia confocal para monitorar a viabilidade por exemplo, diferenciação de células e de alinhamento. Dependendo da aplicação específica, os requisitos para a engineetecido muscular vermelho pode variar; por exemplo, utilização em medicina regenerativa requer o escalonamento do tamanho dos tecidos e a vascularização, enquanto para servir como uma tradução alternativa a outras espécies de carne é necessário.

O produto final será construções musculares, como indicado na Figura 3. O tamanho do tecido será de aproximadamente 8 mm de comprimento, 2 mm de largura e 0,5 mm de espessura. A estimulação eléctrica durante a diferenciação vai alterar a expressão das isoformas de cadeia pesada de miosina, mas não aumentar grandemente o processo de diferenciação, tal como induzida pelo meio de diferenciação 8, mas a estimulação eléctrica também pode ser aplicado no final do processo para verificar a f...

Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Tecido muscular de engenharia tem um grande potencial na medicina regenerativa, como modelo de doença e também como uma fonte alternativa para a carne. Aqui descreve-se a engenharia de uma construção de músculo, neste caso, a partir de células progenitoras, mioblastos de rato e a estimulação por pulsos eléctricos.

Os tecidos músculo esquelético de engenharia a partir de células progenitoras mioblastos murinos e Aplicação da estimulação elétrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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20.8K visualizações.  Eindhoven University of Technology, The Netherlands. Tecido muscular de engenharia tem um grande potencial na medicina regenerativa, como modelo de doença e também como uma fonte alternativa para a carne. Aqui descreve-se a engenharia de uma construção de músculo, neste caso, a partir de células progenitoras, mioblastos de rato e a estimulação por pulsos eléctricos.

Vídeo: Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

Fabricação de Micro-tecidos utilizando Módulos de Colágeno Gel células contendo

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Bioengenharia

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Engenharia baseada em fibrina construções de tecido de miofibroblastos e de aplicação de restrições e tensão para induzir a organização das células e colágeno (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun fibrosa Andaimes de Poli (glicerol-dodecanedioate) para Engenharia Neural tecidos de rato as células estaminais embrionárias

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Medição do isométrica máxima força gerada pelo permeabilizadas fibras musculares esqueléticas

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Atuador baseado em células musculares cardíacas e Biorobot auto estabilizante - PARTE 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

Atuador baseado em células musculares cardíacas e auto-estabilizante Biorobot - Parte 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de microfluidic Bioprinting para a engenharia de tecidos e Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells&#039; Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Estimulação simultânea de elétrica e mecânica para aumentar o potencial de Cardiomyogenic das células

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...