Los autores desean agradecer a Yabin Wu para el cultivo de tejidos que se presentan en la Figura 2, la foto fue tomada por Bart van Overbeeke. El trabajo fue apoyado financieramente por SenterNovem, ISO 42022 donación.

1. Cultura de mioblastos murinos células progenitoras o células C2C12 <ol><li> Aislar las células de acuerdo con el protocolo publicado inicialmente por Shefer y colegas 10 y más tarde adaptado por Collins et al. 11 y Boonen et al. 12 y almacenarlas en nitrógeno líquido. Esto requiere ratones, por ejemplo C57Bl / 6. Los métodos alternativos se utilizan en otros laboratorios, por ejemplo, un método publicado en Journal of experimentos visualizados por Li Y et al. 13. Para los reactivos y el equipo que se hace referencia a la tabla de la página 7 y 8. Desde el músculo de un ratón que generalmente obtener suficientes células para criopreservar aproximadamente 20 viales en nitrógeno líquido. A continuación, se inicia el protocolo de descongelar las células del almacenamiento en nitrógeno líquido. </li><li> Colocar las células de 1 vial en un 25 cm 2 Matrigel (1 mg / ml) matraz de cultivo de tejido recubierto y se añade medio de crecimiento (GM) que consiste en (335 ml DMEM avanzada, 100 ml bovin fetale suero (FBS), 50 ml HS, 5 ml de penicilina / estreptomicina, 5 ml de L-glutamina, 5 ml de extracto de embrión de pollo (CEE)). </li></ol> Nota: la capa por 2 horas a temperatura ambiente y eliminar el Matrigel por aspiración. <ol start="3"><li> Subcultura de las células aproximadamente 1:03 cada 3 días. Esto significa: el día 3: Células paso de un 2 a 25 cm a 75 cm 2 frasco, en el día 6: Células pasaje de un 75 cm 2 frasco a dos 150 cm 2 frascos (con 30 min preplating en frascos sin recubrimiento). El día 9 transferencia de un matraz de 150 cm 2 a 1 triple 150 cm 2 matraz, o si no está disponible a tres matraces de 150 cm 2 (si es necesario la presiembra de nuevo dependiendo del fenotipo de las células). En el día 12 las células están listas para la siembra en una construcción de músculo. </li></ol> Notas: - Por triples 150 cm 2 el número de células será de aproximadamente 4,5 x 10 6 células. - Utilice víals de diferentes aislamientos de mezclarlos para obtener una población mixta de células en el momento de la siembra. Nota: Si decide no trabajar con células primarias, C2C12 mioblastos son una buena alternativa. 2. Ingeniería de tejidos del músculo esquelético <ol><li> Preparar pegamento de silicona mezclando el &quot;elastómero&quot; con el &quot;agente de curado&quot; (10:1). Cortar + / - 5 mm segmentos cuadrados de velcro con una cara triangular (casa-como la forma, la figura 1). Pegamento el Velcro en los pocillos de una placa de cultivo de 6-así en un espacio de aproximadamente 12 mm entre los cuadrados. </li></ol> Notas: - Utilice sólo el lado suave del velcro y la cara hacia arriba el lado este. - Asegúrese de que los tejados se enfrentan entre sí. - Sólo cubrir el velcro con pegamento de silicona, no se extienden a lo largo de la cola también. - Para la estimulación eléctrica posterior es importante hacer coincidir los constructos en dirección vertical del pozo plcomió (a lo largo del eje largo). <ol start="2"><li> Deje secar durante la noche en horno de vacío, no se calienta, principalmente para eliminar burbujas de aire. Esterilizar mediante la adición de 70% de EtOH a los pocillos e incubar durante 15 min. Enjuagar 3 veces con PBS y sometido a UV durante 15 min. Eliminar todo PBS de los pocillos y el Velcro y el lugar en la incubadora hasta su uso. </li><li> Descongelar solución Matrigel en la nevera y preparar la solución de colágeno a la concentración deseada poco antes de hacer los constructos 3D. Se diluye el stock de colágeno con estéril ácido acético 0,02% (concentración final 3,2 mg / ml). </li></ol> Nota: dejar todo en hielo. <ol start="4"><li> Tripsinizar las células, resuspender en GM y contar. Deja células en tubo de centrífuga en la incubadora. </li><li> Agregue la cantidad deseada de solución de colágeno valores para el número de construcciones que quieres hacer a un tubo (de acuerdo a la Tabla 1), añadir 0,5 M de NaOH a la solución de colágeno hasta que una luz colo rosar indica un pH de 7,5. Mezclar suavemente pipeteando arriba y abajo y evitar la formación de burbujas. A continuación, añadir el Matrigel y mezclar muy suavemente pero a fondo. Por último, agregue el GM a la mezcla de colágeno / Matrigel (para las cantidades apropiadas véase el cuadro 1). </li></ol> Notas: - Realice cada paso en el hielo! Matrigel y colágeno fácilmente gel a temperatura creciente. - Cuidado con que el color en realidad es de color rosa! Un aumento de pH también inducir gelificación rápida. - La concentración final de colágeno: 1,6 mg / ml. <ol start="6"><li> Centrifugar la cantidad deseada de las células a 1.000 rpm durante 5 min y eliminar el sobrenadante. </li></ol> Nota: - En función de la actividad de las células el número de células por construir necesario ajustar. Típicamente, el número de células se encuentra entre 750.000 y 1.250.000 células por constructo. <ol start="7"><li> Use un poco de la mezcla de gel para resuspender el pellet celulary transferir las células a la mezcla restante y mezclar a fondo, pero sin introducir burbujas de aire. </li><li> Tomar las placas precalentadas fuera de la incubadora y la pipeta 0,35 -0,4 ml de la mezcla de células-gel en la primera Velcro. A continuación, comienzan a pipetear la mezcla desde el centro entre los sitios de unión y se extienden hasta el Velcro. Por último, utilizar el resto de gel para llenar la brecha entre los dos anclajes de Velcro y la pipeta alrededor de las piezas de velcro. </li><li> Revise cuidadosamente después de 5-10 minutos si los geles son lo suficientemente sólido como para ser transferidos, es decir, golpear suavemente los platos e inspeccionar visualmente si el gel es rígido. Si es así, coloque cuidadosamente los platos en una incubadora. Por lo general, pueden ser recogidos después de diez minutos. Entonces, después de 1-2 horas, suavemente superponer cada gel con 4 ml de GM caliente. </li></ol> Nota:-No haga movimientos vigorosos al manipular las placas. <ol start="10"><li> Reemplace GM por medio de diferenciación (medio de crecimientosin extracto de embrión de pollo) a las 24 h, y sustituirlo por un nuevo medio cada 2-3 días. En el día 7 que debería haber obtenido fibras musculares maduras orientadas, como se puede evaluar con la tinción para el desarrollo estriación transversal de las fibras musculares fusionados. </li></ol> 3. Estimulación eléctrica <ol><li> Esterilizar los electrodos de la placa de ionoptix (véase la tabla de reactivos y equipos) antes de su uso con 70% de etanol y posteriormente se seca bajo UV en una cabina de seguridad. Colocar la placa con unos electrodos sobre la placa de cultivo con las construcciones y cubre con la tapa de la placa de cultivo y la transferencia a una incubadora. Conecte el Ionoptix C-pacer con los cables adecuados. </li></ol> Nota: Los electrodos se colocan en paralelo junto a la construcción de músculo (Figura 2). <ol start="2"><li> Aplicar el protocolo de estimulación como se esperaba. </li></ol> Nota: Por lo general, utilizan 4 V / cm,Pulsos de 6 ms a una frecuencia de 2 Hz. Cambiar medio de cultivo cada 24 horas durante la estimulación.

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Los autores no tienen nada que revelar.

La ingeniería de tejidos musculares tiene un gran potencial para el uso como un modelo de enfermedad, para la selección de fármacos, en medicina regenerativa y la producción de carne. Sin embargo, los requisitos para estas aplicaciones varían. Elegimos trabajar con una combinación de colágeno y Matrigel, colágeno porque permite la alineación celular y porque las células progenitoras de mioblastos requieren la presencia de proteínas de la membrana basal derivados como se determinó en estudios previos de 2D <s...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Matrigel-factor de crecimiento reducido </td><td> Beckton y Dickinson </td><td></td><td></td></tr><tr><td> DMEM (glucosa alta) * </td><td> Gibco </td><td> 42430 </td><td></td></tr><tr><td> Advanced DMEM </td><td> Gibco </td><td> 12491 </td><td></td></tr><tr><td> El suero de caballo </td><td> Gibco </td><td> 65050-122 </td><td></td></tr><tr><td> El suero fetal bovino </td><td> Greiner </td><td> 758075 </td><td></td></tr><tr><td> 0,45 y 0,22 micras filtro de jeringa * </td><td> Whatmann (Schleicher y 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> Cultura plástico </td><td> Greiner </td><td></td><td> Incluye frascos de cultivo y pipetas </td></tr><tr><td> Polluelo extracto de embrión </td><td> Estados Unidos Biológica </td><td> C3999 </td><td></td></tr><tr><td> Pipeta Pasteur * </td><td> Hilgenberg </td><td></td><td> Pipetas Pasteur, con constricción, con algodón, L punta abierta: 230 mm de diámetro con punta de 0,9 - 1,1 mm </td></tr><tr><td> Pipeta Pasteur * </td><td> Hilgenberg </td><td></td><td> Pipetas Pasteur, con constricción, con algodón, L punta abierta: 230 mm de diámetro con punta de 1,4 - 1,6 mm </td></tr><tr><td> Pipeta Pasteur </td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> La colagenasa tipo I* </td><td> Sigma </td><td> C0130-16 </td><td></td></tr><tr><td> 40 micras filtro de células * </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> Aguja 19G </td><td></td><td></td><td></td></tr><tr><td> Elastómero </td><td> Dow Corning Corporation </td><td> 3097358-1004 </td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Agente de curado </td><td> Dow Corning Corporation </td><td></td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Velcro </td><td> Tienda Regular </td><td></td><td> Usted puede comprar en una tienda normal, utilice únicamente el lado suave </td></tr><tr><td> El colágeno tipo I, cola de rata </td><td> BD Biosciences </td><td> 3544236 </td><td></td></tr><tr><td> C-Pace EP Pacer Cultura </td><td> Ionoptix </td><td></td><td></td></tr><tr><td> 6-y las placas de cultivo para la estimulación eléctrica </td><td> Beckton Dickinson-Falcon </td><td> BD Falcon # 353846 </td><td></td></tr><tr><td> Dish C-electrodos cultura plato </td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> * Necesaria para el aislamiento de células (punto 1,1) # Juntos en un kit </td></tr></tbody></table>

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

Engineered tejidos musculares se pueden utilizar para diferentes propósitos, que incluyen la producción de tejidos para su uso como un modelo de enfermedad in vitro, por ejemplo, para estudiar las úlceras por presión, para la medicina regenerativa y como una alternativa de la carne 1. Las primeras construcciones del músculo informaron 3D se han hecho desde hace muchos años y pioneros en el campo son Vandenburgh y sus colegas 2,3. Los avances en la ingeniería de los tejidos musculares no son sólo el resultado de la ganancia enorme en el conocimiento de los factores bioquímicos, células madre y células progenitoras, pero en especial basada en los conocimientos adquiridos por los investigadores que los factores físicos juegan un papel esencial en el control del comportamiento de las células y tejido de desarrollo. State-of-the-art músculo ingeniería construye actualmente consisten en construcciones de hidrogel de células pobladas. En nuestro laboratorio éstas generalmente constan de células mioblastos murinos progenitoras, aislado de músculos de las extremidades traseras murinos o una línea celular de mioblastos de ratón C2C12, millasjado con una mezcla de colágeno / Matrigel y se sembraron entre dos puntos de anclaje, imitando los ligamentos musculares. Otras células pueden ser considerados también, por ejemplo, líneas celulares alternativas tales como mioblastos de rata L6 4, 5 neonatales musculares derivadas de células progenitoras, células derivadas de tejidos musculares adultos de otras especies, tales como humano 6 o incluso células madre pluripotentes inducidas (células iPS) 7 . Contractilidad celular provoca la alineación de las células a lo largo del eje largo de la construcción de 8,9 y la diferenciación de las células progenitoras de músculo después de aproximadamente una semana de cultivo. Además, la aplicación de la estimulación eléctrica puede mejorar el proceso de diferenciación, hasta cierto punto 8. Debido a su tamaño limitado (8 x 2 x 0,5 mm) del tejido completa se pueden analizar utilizando microscopía confocal para controlar la viabilidad por ejemplo, la diferenciación celular y la alineación. Dependiendo de la aplicación específica de los requisitos para la ingetejido muscular rojo variarán, por ejemplo, uso para la medicina regenerativa requiere la ampliación de tamaño del tejido y la vascularización, mientras que para servir como una traducción alternativa de la carne de otras especies es necesario.

El producto final será constructos musculares, como se indica en la Figura 3. El tamaño del tejido será de aproximadamente 8 mm de largo, 2 mm de ancho y 0,5 mm de espesor. La estimulación eléctrica durante la diferenciación cambiará la expresión de las isoformas de cadena pesada de miosina, pero no mejoran en gran medida el proceso de diferenciación inducida por el medio de diferenciación 8, pero la estimulación eléctrica se puede aplicar también al final del proceso para comprobar la func...

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

Tejido muscular Engineered tiene un gran potencial en medicina regenerativa, como modelo de la enfermedad y también como una fuente alternativa para la carne. Aquí se describe la ingeniería de una construcción de músculo, en este caso a partir de células progenitoras de mioblastos de ratón y la estimulación por impulsos eléctricos.

Los tejidos de ingeniería músculo esquelético de células mioblastos murinos progenitoras y aplicación de estimulación eléctrica

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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Research

JoVE Journal

Bioengineering

20.9K vistas.  Eindhoven University of Technology, The Netherlands. Tejido muscular Engineered tiene un gran potencial en medicina regenerativa, como modelo de la enfermedad y también como una fuente alternativa para la carne. Aquí se describe la ingeniería de una construcción de músculo, en este caso a partir de células progenitoras de mioblastos de ratón y la estimulación por impulsos eléctricos.

Video: 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.

Fabricación de micro-tejidos, utilizando los módulos de gel de colágeno las células que contienen

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

Investigación

Bioingeniería

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.

Ingeniería fibrina basado en construcciones de tejido de miofibroblastos y de aplicación de las restricciones y la tensión para inducir la organización de células y 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 fibroso Andamios de poli (glicerol-dodecanodioato) de Ingeniería Neural Los tejidos de las células madre embrionarias de ratón

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.

Medición de la fuerza isométrica máxima generada por las fibras musculares esqueléticas permeabilizadas

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

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.

Actuador basado en células musculares cardíacas y biorobot autoestabilizante - 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.

Actuador basado en las células musculares cardíacas y 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 Bioprinting de microfluidos para la ingeniería de tejidos y organitas

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.

Dos métodos de descelularización de tejidos vegetales para aplicaciones de ingeniería de tejidos

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.

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.

Simultánea estimulación eléctrica y mecánica para mejorar el potencial de las células cardiomiogenético

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

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Construcción y uso de una cámara de estimulación eléctrica para mejorar la diferenciación osteogénica de células madre mesenquimales, estromales In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...