Los autores desean agradecer NSERC Discovery Grant 402462 &quot;andamiaje de tejido de ingeniería para el control de células madre pluripotentes inducidas comportamiento de las células&quot;.

Notas antes de iniciar el protocolo: El fibrinógeno es una proteína de la sangre y la formación derivada de seguridad tanto, conviene que debe ser completado antes de su uso. Este protocolo requiere de 2 días para completar lo que el tiempo de las culturas madre deseadas apropiada para asegurarse de que están listos para la siembra. En términos de cálculo de la cantidad de fibrinógeno para pesar, tres 35 mm placas de Petri de solución salina tamponada con Tris (TBS, pH 7,4) que contenía 110-130 mg de fibrinógeno disuelto en 3 ml de TBS será suficiente para producir una placa de 24 pocillos que contenía 400 andamios l de fibrina en cada pocillo. 1. Día Uno: Hacer las soluciones de fibrinógeno y de diálisis durante la noche <ol><li> Tome fibrinógeno liofilizado de la nevera y dejar que repose durante 20 minutos para permitir que llegue a temperatura ambiente. </li><li> Añadir 3 ml de solución salina tamponada con Tris (TBS) a cada 35 mm de placa de Petri. Cada placa de los rendimientos de fibrinógeno 2 a 3 ml de solución de fibrinógeno que van en la concentración de 12 a 20mg / ml y la cantidad total de fibrinógeno es necesario se puede calcular sobre la base de estas aproximaciones. </li><li> Pesar aproximadamente 100-130 mg de fibrinógeno (Fg) y rociar sobre la superficie de la presente TBS en cada plato. Espere 5 minutos para que el fibrinógeno para empezar a ir a la solución. Cubrir el plato de Petri con tapa. </li><li> Incubar las placas Petri a 37 ° C durante 2 horas para permitir que el fibrinógeno a ir completamente en la solución de TBS. </li><li> Tubos de diálisis Wet (7000 MW de corte) con TBS. Doble el extremo inferior del extremo del tubo y la pinza se cerró con una pinza de diálisis. </li><li> Pipetear las soluciones de fibrinógeno de los platos en tubos de diálisis. Dobla el extremo superior y la abrazadera de cierre con una pinza de diálisis. </li><li> Colocar tubo de diálisis que contiene solución de fibrinógeno en un recipiente 4 litros de TBS. Mezcle la solución con placa de agitación ajustado a baja velocidad (100 rpm). </li><li> Dializar solución durante la noche en agitar la placa por lo menos 12 horas. La solución de TBS no necesita ser cambiado durante este período de tiempo. </li></ol&gt; 2. Día 2: La polimerización de los andamios de fibrina y la siembra de células madre en los andamios Todos los pasos descritos para este proceso debe llevarse a cabo en una campana de cultivo de tejidos estéril como estos andamios se sembraron con cultivos de células madre. <ol><li> Retire la solución de fibrinógeno en tubos de diálisis y el lugar en un tubo de tamaño apropiado cónica (o bien 15 mL o 50 mL). </li><li> Solución de fibrinógeno de filtro con un filtro de 5,0 micras jeringa para eliminar las impurezas grandes en la solución. Dependiendo del volumen de la solución, el uso de múltiples filtros pueden ser necesarias para procesar toda la solución. </li><li> Filtro estéril del fibrinógeno con solución de 0,22 micras de filtro en un tubo de 50 ml cónico. Utilizando una pipeta tamaño apropiado, determinar el volumen de la solución de fibrinógeno se filtró para utilizar más adelante al hacer cálculos. Dependiendo del volumen de la solución, el uso de múltiples filtros puede ser necesario para procesar la ENTla ira de solución. </li><li> Medir la absorbancia de la solución de fibrinógeno a 280 nm utilizando un espectrofotómetro. Un factor de dilución de 50 veces es necesario obtener una absorbancia bajo 1. </li><li> Calcular la concentración de proteína presente en la solución de fibrinógeno utilizando la siguiente ecuación: [fibrinógeno en mg / ml] = (A x 280 factor de dilución) ÷ 1,55 donde 1,55 es el coeficiente de extinción a un 280 por fibrinógeno humano 13. A continuación calcular la cantidad de TBS necesaria para diluir la concentración de la solución a 11,1 mg / ml de fibrinógeno en base al volumen inicial. La concentración final de proteína en la fibrina andamios será de 10 mg / ml después de la polimerización. </li><li> Obtener trombina estéril y soluciones de cloruro de calcio. En primer lugar, añadir 15 l solución de trombina (concentración: 40 U / ml - concentración final en andamio será de 2 U / ml) al lado derecho de cada pocillo en la primera fila de la placa de 24 pocillos. A continuación, añadir 15 l de 50 mM de calcio chloride solución al lado izquierdo de cada pocillo. Finalmente, añadir 270 l de la solución de fibrinógeno a la placa. Agitar la placa de lado a lado seguido por agitación de atrás hacia delante para asegurar el mezclado de las soluciones. </li><li> Que la fila que contiene las mezclas polimerizar durante 5 minutos a temperatura ambiente. Los armazones se hacen más opaca a medida que se polimerizan. </li><li> Repetir los pasos 2.6 y 2.7 hasta que el número deseado de andamios fibrina han sido polimerizado. </li><li> Después de la incubación último minuto 5, colocar las placas dentro de una incubadora a 37 ° C para permitir la polimerización completa de andamios. </li><li> Después de la incubación de una hora es completa, el siguiente paso es la semilla del andamio con cuerpos embrioides. El uso de un 20 pipettemen l, seleccionar un organismo de embrioide individuo y su lugar en el centro del andamio de fibrina. Comprobar con el microscopio que cada pozo tiene un cuerpo embrioide sola. </li><li> Añadir 5 l de solución de trombina (concentración: 40 U / ml) y 5 l de cloruro de calcio 50 mMsolución en la parte superior de cada cuerpo embrioide seguido por 90 l de solución de fibrinógeno a cada pocillo. La solución debe formar una burbuja encapsular los cuerpos embrionarios. </li><li> Colocar la placa posterior en incubadora a 37 ° C durante una hora adicional para asegurar la polimerización. </li><li> Después de la incubación, se añade 1 ml de los medios de cultivo celulares apropiadas a cada pocillo de placa y el lugar de nuevo en 37 ° C incubadora. </li></ol> 3. Los resultados representativos La figura 1 muestra una vista esquemática de la vista lateral de un individuo, así que contiene el sistema de cultivo 3D fibrina andamio sembró con un cuerpo embrioide. Figura 2A muestra imágenes representativas de ratón inducido células madre pluripotentes siendo cultivados en fibroblastos de embriones de ratón capas alimentadoras. Estas células son luego inducidas a formar cuerpos embrionarios, los agregados de células que contienen células progenitoras neurales, utilizando un ácido retinoico 8 días el protocolo de tratamiento (Figura 2B) como previously descrito 14 mientras que la Figura 3 muestra el aspecto de una iPS derivado cuerpo embrioide después de 3 días de cultivo en el interior de andamios 3D fibrina. Resultados similares se han obtenido anteriormente con células madre embrionarias de ratón 7. Además, este método ha sido usado para cultivar IPS-derivados cuerpos embrioides producido utilizando un protocolo diferente que implica ácido retinoico y purmorphamine 15 dentro de andamios 3D fibrina, lo que demuestra la versatilidad de este sistema de cultivo. <img alt="Figura 1." src="/files/ftp_upload/3641/3641fig1.jpg" /> Figura 1. Esquemática que muestra la vista lateral de un individuo, así que contiene un andamio 3D fibrina sembrado con un cuerpo embrioide. <img alt="Figura 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> Figura 2. Ratón madre pluripotentes inducidas (iPS) de cultivo celular y la diferenciación. Para diferenciar estas células en neurfenotipos al, las células iPS se cultivan en suspensión para producir agregados de células llamadas cuerpos embrioides (EBS). Estos EBs son tratados con ácido retinoico para inducir la diferenciación neuronal y este protocolo se utilizó anteriormente para inducir la diferenciación neural de células madre embrionarias. A) del ratón colonia indiferenciada de células iPS cultivadas en embriones de ratón capa alimentadora de fibroblastos. B) cuerpos embrionarios derivados de las células iPS de ratón en cultivo en suspensión tomada el día 8 después del tratamiento con ácido retinoico para inducir la diferenciación neuronal. Barra de escala es de 100 m. <img alt="Figura 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> Los resultados de la Figura 3. Ejemplo de una iPS de ratones embrionarios cuerpo después de 3 días de cultivo en el interior de una estructura 3D de fibrina. Las células han comenzado a migrar y diferenciarse en el interior del andamio de fibrina. Barra de escala es de 500 m.

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Los autores no tienen nada que revelar.

Este protocolo detallado anteriormente proporciona un método para generar 3D andamios fibrina para el cultivo de células madre pluripotentes, específicamente para embriones de ratón y células madre pluripotentes inducidas. Este sistema 3D biomaterial cultura basada más reproduce con precisión el nicho de células madre encontradas in vivo y, como resultado, se puede utilizar para rastrear las señales biológicas para determinar sus efectos sobre la diferenciación de células madre 6.</sup...

Equipo necesario: Balanza analítica pH metro Cultivo de tejidos incubadora (37 ° C, 5% de CO 2) Revuelva la placa Espectrofotómetro Tejido estéril campana de la cultura Tris solución salina tamponada (pH 7,4) (Need 4 L más suficiente para disolver el fibrinógeno) 50 mM de solución de cloruro de calcio Estériles tubos cónicos (15 o 50 ml) 35 mm placas de Petri Diálisis tubo (7.000 MW de corte) Clips de diálisis 5.0 micras filtros de jeringa Envueltas individualmente estériles filtros de 0,22 micras de jeringa Jeringa 24 placas de cultivo tisular <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> El fibrinógeno (humano) </td><td> Calbiocdobladillo </td><td> 341578 </td><td></td></tr><tr><td> La trombina (humano) </td><td> Sigma </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

Las células madre se encuentran en forma natural microambientes 3D in vivo, que se refieren a menudo como el nicho de células madre 1. El cultivo de células madre en el interior de 3D ​​biomateriales proporciona una manera de imitar con precisión estos microambientes, proporcionando una ventaja sobre los métodos tradicionales de cultivo 2D utilizando poliestireno, así como un método para tejidos de reemplazo de ingeniería 2. Mientras poliestireno 2D cultivo de tejidos se ha utilizado para la mayoría de los experimentos de cultivo celular, 3D biomateriales puede replicar más estrechamente los microambientes encontrados in vivo, permitiendo establecimiento más preciso de la polaridad celular en el medio ambiente y que posee propiedades bioquímicas y mecánicas similares a los tejidos blandos. 3 Una variedad de de origen natural y sintéticos biomateriales han sido investigados como entornos 3D para apoyar el crecimiento de células madre. Mientras andamios sintéticos pueden ser sintetizados a tener una mayor range de propiedades mecánicas y químicas y, a menudo tienen una mayor reproducibilidad, biomateriales naturales a menudo están compuestos de proteínas y polisacáridos que se encuentran en la matriz extracelular y, como resultado contienen sitios de unión para la adhesión celular y fácilmente apoyar el cultivo de células. Andamios fibrina, producido por la polimerización del fibrinógeno proteína obtenida a partir de plasma, se han investigado ampliamente para una variedad de aplicaciones de ingeniería de tejidos, tanto in vitro como in vivo 4. Tales andamios se pueden modificar mediante una variedad de métodos para incorporar los sistemas de liberación controlada para la entrega de factores terapéuticos 5. Trabajos previos han demostrado que tales andamios puede ser utilizado con éxito para cultivar células madre embrionarias y este sistema de cultivo basado en andamio puede ser utilizado para cribar los efectos de diversos factores de crecimiento sobre la diferenciación de las células madre sembradas en el interior 6,7. Este protocolo detalles el proceso de polymerizing fibrina andamios a partir de soluciones de fibrinógeno utilizando la actividad enzimática de la trombina. El proceso de toma de 2 días para completar, incluyendo una etapa de diálisis durante la noche para la solución de fibrinógeno para eliminar citratos que inhiben la polimerización. Estos métodos se basan en detalle las concentraciones de fibrinógeno decididos a ser óptimo para el cultivo de células pluripotentes embrionarias y pluripotentes. Otros grupos han seguido investigando andamios fibrina para una amplia gama de tipos de células y aplicaciones - demostrar la versatilidad de este enfoque 8-12.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Estos detalles del trabajo de la preparación de la fibrina andamios 3D para el cultivo y diferenciación de las células madre plutipotent. Tales andamios puede ser utilizado para cribar los efectos de diversos compuestos biológicos sobre el comportamiento de células madre, así como modificado para contener los sistemas de suministro de fármacos.

Preparación de fibrina Andamios 3D para Aplicaciones madre para cultivo celular

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

preparation-of-3d-fibrin-scaffolds-for-stem-cell-culture-applications

Research

JoVE Journal

Bioengineering

25.1K vistas.  University of Victoria. Estos detalles del trabajo de la preparación de la fibrina andamios 3D para el cultivo y diferenciación de las células madre plutipotent. Tales andamios puede ser utilizado para cribar los efectos de diversos compuestos biológicos sobre el comportamiento de células madre, así como modificado para contener los sistemas de suministro de fármacos.

Video: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Investigación

Bioingeniería

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Preparación de la muestra Estrategias para Espectrometría de Masas de imágenes de modelos de cultivo celular en 3D

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D hidrogel andamios para articular condrocitos cultura y cartílago Generación

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabricación de Mecánicamente sintonizable y Bioactivos metal andamios para Aplicaciones Biomédicas

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning y Montaje de Microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Síntesis de biocompatibles de cristal líquido de elastómero Espumas como andamios celulares para 3D espaciales Cultivos Celulares

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Construcción de una hoja de varias capas células madre mesenquimales con un sistema de cultivo dinámico 3D

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Un método de cultivo para mantener las células madre hematopoyéticas humanas quiescentes

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Preparación y caracterización de biotinta de hidrogel biohíbrida 3D basada en grafeno para neuroingeniería periférica

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...