Los autores agradecen a Neil Whitehouse (WPI, Tienda Higgins de la máquina) por su ayuda con el mecanizado CNC. Además, nos gustaría agradecer a Adriana Hera (WPI Informática y el Centro de Comunicaciones) por su ayuda en MATLAB de programación, así como bebidas Kate y José Cotnoir (WPI Académico del Centro de Tecnología) para obtener ayuda con Camtasia. Sophie Burke y Becker Jacleen (WPI Académico del Centro de Tecnología) proporciona imágenes de vídeo adicional. Este trabajo fue financiado por los Institutos Nacionales de Salud (R15 HL097332), la Universidad de Massachusetts Medical School-IPM iniciativa piloto de investigación, la American Heart Association (beca de investigación de pregrado a JZH), y el Instituto Politécnico de Worcester (Becas de Verano de Investigación de Pregrado a JZH e institucional la puesta en marcha de fondos para MWR).

1. La siembra de células de fabricación de moldes Comience por el fresado de una 1 / 2 &quot;pedazo grueso de policarbonato para crear 15, de fondo redondo, pozos anular con un diámetro de poste central de 2 mm. Los canales de fresado de 6 mm de profundidad y 3,75 mm de ancho. Limpie y seque el molde de policarbonato eliminar los residuos plásticos del proceso de molienda. Mezcla de polidimetilsiloxano (PDMS) en una proporción de 10:1 (w / w) de la base de que el agente de curado, desgasificar para eliminar todas las burbujas de aire, y se vierte en el molde de policarbonato. Degas de nuevo para eliminar las burbujas restantes, y la curación en el horno a 60 ° C durante 4 horas. Una vez curado, retire con cuidado la plantilla de PDMS lentamente separándola de la de policarbonato, lavar con agua y jabón, y autoclave. También autoclave una solución de agarosa dos por ciento (w / v) disuelto en medio Dulbecco modificado por Eagle (DMEM). Coloque la plantilla de PDMS sobre una superficie plana y se llenan de líquido aga<html> aumentó en un primero de agarosa pipetear en cada uno de los moldes centro de mensaje, a continuación, poniendo en el vaso espacio a su alrededor. Deje que la agarosa a solidificar (15 minutos), luego invierta el molde para liberar la agarosa de PDMS. Corte el exceso de agarosa de todo cada uno de los pozos, y el lugar de los pozos de agarosa en placas de 12 pocillos. 2. Cultivo de células y la siembra de anillo Añadir celular medios de cultivo (DMEM con FBS al 10% y 1% de penicilina-estreptomicina) en el exterior del molde de agarosa, sin cubrir la parte superior de los moldes. Coloque las placas en la incubadora y les permite equilibrar durante aproximadamente 15 minutos mientras prepara las células. A 90% de confluencia, trypsinize células del músculo liso de aorta de rata (CMRE) y volver a suspender a una concentración de 5x10 6 células / ml. Pipetear 100 L de la suspensión celular en cada uno de los pozos de agarosa al anular, mediante un movimiento circular para aplicar las células a cada pocillo. contenido &quot;&gt; Asegúrese de que los estantes incubadora están al mismo nivel, a continuación, colocar las placas en la incubadora y les permite sentarse en reposo durante 24 horas. Cambio de medio cada dos días después por aspiración de los medios de comunicación de todo el bien, y volver a llenar y cada uno hasta que el agarosa y está completamente sumergida. 3. Tejido de cosecha del anillo y la medición de espesores Al término de la cultura, retirar el anillo del molde de agarosa al deslizar sobre la parte superior del poste central. Coloque el anillo en una pequeña placa de Petri con tampón fosfato salino (PBS), el centro de la muestra, y adquirir una imagen utilizando un sistema de imagen digital. Use un programa de análisis de imágenes para la detección de bordes y medir el grosor del anillo en 4 posiciones (superior, inferior, izquierdo y derecho) alrededor de su circunferencia. Calcular el valor de grosor medio (t) para cada anillo individual, y utilizar este valor para calcular el área de sección transversal(2π (t / 2) 2). 4. Las pruebas mecánicas de los anillos Configure la máquina de tracción (Instron EPS 1000) en la posición horizontal. Adjunte una célula de carga ± 1N 1mN y la costumbre apretones de alambre delgado (creado por la flexión de alambre de acero inoxidable). Montar la muestra de anillo en las dos asas de alambre delgado. Se extienden las garras hasta que un 5 mN carga de tara se aplica a la muestra. Entrar en el área de sección transversal (calculado a partir de las mediciones de espesor) y registrar la longitud de referencia. Pre-ciclo de los anillos de 8 veces entre la carga de tara y 50 kPa tensión a una velocidad de 10 mm / min. Después de la octava pre-ciclo, tirar al fracaso a los 10 mm / min. Si no se observa como una disminución de la fuerza en un 40% de una medición a la siguiente. Para cada muestra, el anillo, medir la carga última (UTS) y la tensión y la falta calcular el máximo módulo tangente (MTM) de los datos adquiridos. 5. Tejidos como el anilloAsamblea para la fabricación de tubos de tejido Los titulares de costumbre están fabricadas en tubo de discos redondos de 5 cm de policarbonato con cortes rectangulares (2 cm x 3 cm). Perforar los agujeros roscados a través de estos discos para permitir que dos titulares que se atornillan. Autoclave de los titulares de tubo de costumbre, y luego se transfieren a la cabina de bioseguridad y el lugar en un vacío placa de Petri. Use tijeras quirúrgicas para cortar los extremos de 1,9 mm de diámetro, los tubos de silicona en un ángulo para crear bordes biselados, autoclave y luego los tubos de silicona. Mientras que los tubos están en autoclave, llenar una caja de Petri con medios de comunicación. Quitar los anillos de los pozos de agarosa y se colocan en la placa de Petri con medios de comunicación. Lugar autoclave tubos de silicona en la misma placa de Petri para humedecerlos antes de su uso. Use unas pinzas para colocar un extremo biselado de un tubo de silicona en el centro de un anillo y deslice suavemente el anillo en el tubo de silicona. Repita el procedimiento con el número deseado de riNGS. Deslice los anillos en contacto unos con otros con suavidad empujando sucesivamente en ambas direcciones a lo largo del tubo. Este método puede ser utilizado para los anillos de cosecha en un solo día en la cultura. Una vez que los anillos se montan, alinear los tubos de silicona dentro de los titulares de policarbonato y el tornillo de las dos partes del titular juntos. Coloque el soporte en una placa de Petri de 100 mm y agregar 55 ml de medio. Intercambio de los medios de comunicación cada 3 días durante la duración de la cultura. Para quitar los tubos de los tejidos, la liberación de los tubos de silicona de la titular de policarbonato y el uso de fórceps para deslizar los tubos de tejido del tubo de silicona y en una placa de Petri llena de PBS. 6. Los resultados representativos: Cuando el protocolo se realiza correctamente, las células se agregan para formar los anillos de tejido con un diámetro interior igual al diámetro del molde correspondiente mensaje dentro de las 24 horas. Los bordes del anillo son generalmente lisas y en apariencia (si es culturalmenteed en una superficie plana), son uniformes en espesor en toda la circunferencia. Los anillos de los tejidos son fáciles de manejar y pueden ser removidos de sus pozos para su posterior análisis histológico y mecánica (ver en Gwyther et al., 2011 8). Morfología de los tejidos del anillo, composición de la matriz, y las propiedades mecánicas varían en función del número y tipo de células sembradas. Los resultados representativos de los anillos de tejido a partir de diferentes tipos de células, las condiciones de siembra, y la longitud de la cultura se muestran en la Tabla 1. Dos anillos mm de diámetro creado a partir de 5x10 5 CMRE exhibió una UTS superiores a los anillos creados a partir de nuestra publicado anteriormente dos anillos mm (a partir de 6.6x10 5 CMRE). Al igual que las células de rata, las células del músculo liso (hSMC) 8 fácilmente agregados para formar tejidos anillos, que contenía una gran cantidad de colágeno después de sólo 14 días de cultivo (datos no mostrados). Las células madre mesenquimales (hMSC) también agregan y forman anillos de cohesión, pero lacked fuerza mecánica y se rompió durante las fases iniciales de las pruebas de precycling tracción uniaxial. <table border="1"><tbody><tr><td></td><td> n </td><td> El número de células </td><td> Espesor (mm) </td><td> Duración de la cultura (días) </td><td> UTS (kPa) </td><td> MTM (kPa) </td><td> Si no la tensión (mm / mm) </td></tr><tr><td> CMRE de los anillos </td><td> 6 </td><td> 660000 </td><td> 0,94 ± 0,12 </td><td> 14 </td><td> 97 ± 30 </td><td> 497 ± 91 </td><td> 0,50 ± 0,08 </td></tr><tr><td> CMRE de los anillos </td><td> 4 </td><td> 500000 </td><td> 0,53 ± 0,02 </td><td> 7 </td><td> 113 ± 8 </td><td> 189 ± 15 </td><td> 0,88 ± 0,05 </td></tr><tr><td> hSMC anillos </td><td> 3 </td><td> 750000 </td><td> 0,51 ± 0,05 </td><td> 14 </td><td> 160 ± 30 </td><td> 270 ± 20 </td><td> 0,92 ± 0,08 </td></tr><tr><td> hMSC anillos </td><td> 3 </td><td> 750000 </td><td> 0,40 ± 0,07 </td><td> 14 </td><td> N / A </td><td> N / A </td><td> N / A </td></tr></tbody></table> Tabla 1. La tabla muestra los parámetros de la cultura y las propiedades mecánicas de dos anillos mm generados a partir de diferentes tipos de células, las concentraciones de la siembra, y la duración de la cultura. <img alt="Figura 1" src="/files/ftp_upload/3366/3366fig1.jpg" /> Figura 1. (A) Esquema del proceso de generación de tejido del anillo. (B) Personal de policarbonato molde con fresado anular los pozos. Diámetro central de correos de 2 mm. (C) PDMS plantilla después de que se despega del molde de policarbonato. (D) anillo de tejido agregado cultivadas en un molde de agarosa con un puesto de 2 mm. (E) de diámetro Dos mm anillo de tejido en PBS. Las barras de escala = 6 mm (B, C) y 2 mm (D, E). <img alt="Figura 2" src="/files/ftp_upload/3366/3366fig2.jpg" /> Figura 2. Trama Representante de la curva de productores de tensión-deformaciónced de ensayo de tracción uniaxial.

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No hay conflictos de interés declarado.

Recientemente, ha habido un creciente interés en la base de células o &quot;andamio-menos&quot; los métodos de la ingeniería de tejidos para hacer frente a algunas de las limitaciones de los enfoques basados ​​en los andamios de ingeniería de tejidos. Dado que células derivadas de tejidos creados a partir de las células y la matriz que producen, que sí contienen mucho mayor densidad de células, no contienen materiales exógenos, y se puede hacer todo a partir de células humanas y de las proteínas. Injertos vasculares a partir de células humanas pueden alcanzar una resistencia mecánica considerable en la ausencia de andamios exógenos (por ejemplo, la presión de ruptura 3400 mmHg en comparación con 1600 mm de Hg para la vena safena humana) 12. Aunque basadas en células tejidos vasculares muestran mejoría en la densidad celular y la resistencia mecánica, métodos de fabricación más reciente (como &quot;hoja de ingeniería con sede en&quot; 3,4,12 o &quot;bioprinting&quot; 5,13) requieren largos períodos de cultivo (&gt; 3 meses) o equipos especializados para la construcción de tejido 3D. El derivado de las células del tejido del anillo de metanfetaminaod descrito aquí permite una rápida celular de auto-ensamblaje para formar sólidas construcciones de tejido en 3D en un plazo corto de tiempo y sin el uso de equipo especializado. Este protocolo se detalla el procedimiento que hemos desarrollado la creación de 2 mm de células derivadas de diámetro interior rata anillos de músculo liso de tejidos. En el presente ejemplo, los anillos de los tejidos se cultivaron durante 7 días (luego cultivaron durante 7 días adicionales para la fusión del anillo y la formación del tubo). Sin embargo, dos ratas mm (y humanos) de las células musculares lisas anillos pueden ser retirados de los pozos y son lo suficientemente coherente para el manejo (por ejemplo, transferir a tubos de silicona) tan pronto como un día después de la siembra de células. Además, los anillos de tejido sólido con distintos diámetros interiores (2, 4 y 6 mm) se pueden crear con este método, simplemente cambiando el diámetro del poste de la original del molde de policarbonato. 8 Recientemente también modificó el diseño de moldes de policarbonato para permitir que cinco de 2 pozos mm de sembrar para ser emitidos en una sola cámara multi-y agarosa, queutiliza menos PDMS y agarosa, y cabe en un pocillo de una placa de 6 pocillos (datos no mostrados). Los cambios en el diámetro del poste, el ancho de la siembra, así, el radio de curvatura de la parte inferior redondeada, el número de pozos de la siembra, o la profundidad de los pozos de siembra pueden ser modificados con sólo cambiar las especificaciones en el archivo de CAD para el CNC mecanizado del molde de policarbonato. Finalmente, un solo molde de policarbonato puede ser utilizado para fabricar un número ilimitado de plantillas de PDMS y PDMS cada plantilla se puede limpiar, autoclave y reutilizado docenas de veces. Además de cambiar el tamaño de los anillos de tejido, hemos hecho los anillos de muchos tipos de células diferentes, incluyendo: SMC primarios de rata (aplicaciones de células, R354-05), principal SMC humanos la arteria coronaria (Lonza, CC-2583), la enseñanza primaria humanos fibroblastos dérmicos 11 (generoso regalo del doctor George Pines, WPI Departamento de Ingeniería Biomédica), fibroblastos de pulmón de rata (RFL-6, ATCC CCL-192), y las células madre mesenquimales (Lonza, PT-2501). Cada uno de estos tipos de células agregados y contratos de todo el centro de mensajes para formar anillos de tejido, aunque la organización celular, la composición de ECM, y las propiedades mecánicas de las construcciones varían para cada tipo de célula. Los parámetros de siembra para cada tipo de célula debe ser determinada empíricamente en función del tamaño de las células y su capacidad de agregar. Por lo tanto, si bien este sistema de creación de células derivadas de los anillos de tejido es extremadamente versátil, el protocolo puede necesitar pequeños ajustes para la formación de tejido óptima con diferentes tipos de células. La geometría del tejido del anillo facilita una fácil carga y evaluación de las propiedades de los materiales de tejido de ensayo de tracción uniaxial, como se describe. También hay precedentes importantes para el uso de sangre de segmentos de anillos vaso para medir la contracción vascular y la función fisiológica. Estudios preliminares indican que células derivadas de los anillos de tejido puede ser montado en un dispositivo de miografía de alambre para la medición de la respuesta farmacológica ygeneración de la fuerza contráctil (datos no mostrados). En total, la capacidad de fabricar rápidamente auto-ensamblado de anillos de células para el análisis histológico, mecánicos, fisiológicos, bioquímicos y sugiere una nueva y poderosa herramienta que puede ser útil para el modelado de la estructura del tejido vascular y la función de la salud y la enfermedad.

<table border="1"><tbody><tr><th> Nombre del reactivo / equipo </th><th> Empresa </th><th> Catálogo # </th></tr><tr><td> Polidimetilsiloxano (PDMS) </td><td> Dow Corning </td><td> Sylgard 184 </td></tr><tr><td> Agarosa </td><td> Lonza </td><td> 50000 </td></tr><tr><td> DMEM </td><td> Mediatech </td><td> 15 a 017 CV </td></tr><tr><td> De suero fetal bovino (FBS) </td><td> PAA </td><td> A05-201 </td></tr><tr><td> Penicilina / estreptomicina </td><td> Mediatech </td><td> 30 a 002-CI </td></tr><tr><td> Sistema de imagen digital </td><td> TVP Corporación </td><td> Modelo 630 </td></tr><tr><td> Máquina de ensayo uniaxial </td><td> Instron </td><td> ElectroPuls E1000 </td></tr><tr><td> Software de detección de bordes </td><td> TVP Corporación </td><td> Marco 2.4.6 </td></tr></tbody></table>

directed cellular self-assembly to fabricate cell-derived tissue rings for biomechanical analysis and tissue engineering

Cada año, cientos de miles de pacientes se someten a cirugía de revascularización coronaria en los Estados Unidos. 1 Aproximadamente un tercio de estos pacientes no han adecuado los buques cedentes autólogo debido a la progresión de la enfermedad o de la cosecha anterior. El objetivo de la ingeniería de tejidos vasculares es desarrollar una fuente alternativa adecuada para estos injertos. Además, la ingeniería tisular vascular pueden resultar útiles como modelos vivos vascular para estudiar las enfermedades cardiovasculares. Varios enfoques prometedores para los vasos sanguíneos de ingeniería han sido exploradas, con muchos estudios recientes centrados en el desarrollo y el análisis de los métodos basados ​​en células. 2-5 Aquí, presentamos un método para rápidamente se auto-ensamblan las células en los anillos de tejido 3D que se pueden utilizar en modelo in vitro para los tejidos vasculares. Para ello, las suspensiones de células de músculo liso se siembran en pocillos de fondo redondo anular agarosa. Las propiedades no adhesivo de la agarosa permitir ªlas células e para resolver, agregar y contrato de alrededor de un poste en el centro del pozo para formar un anillo de tejido cohesionado 6,7. Estos anillos pueden ser cultivadas durante varios días antes de la cosecha para la mecánica, el análisis fisiológicos, bioquímicos o histológicos. Hemos demostrado que estos anillos de tejidos derivados de células rendimiento a 100-500 kPa fuerza a la tracción 8, que excede el valor reportado para otras construcciones de ingeniería tisular vascular cultivadas por períodos similares (&lt;30 kPa). 9,10 Nuestros resultados demuestran que la célula robusta derivados de la generación de tejido vascular del anillo se puede lograr en un corto período de tiempo, y ofrece la oportunidad para la evaluación directa y cuantitativa de los aportes de las células y la matriz de células derivadas de Desarrollo Limpio (MDL) de la estructura del tejido vascular y la función.

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Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Este artículo describe un método versátil para crear células derivadas de los anillos de tejido celular por auto-ensamblaje. Las células musculares lisas sembradas en forma de anillo total de agarosa pozos y el contrato de forma sólida en tres dimensiones (3D) de los tejidos dentro de 7 días. A escala milimétrica los anillos de tejido son propicias para los ensayos mecánicos y servir de base para el montaje del tejido.

Dirigida celular de auto-ensamblaje para fabricar células derivadas de tejido para el análisis de anillos de Biomecánica e Ingeniería de Tejidos

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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18.8K vistas.  Worcester Polytechnic Institute. Este artículo describe un método versátil para crear células derivadas de los anillos de tejido celular por auto-ensamblaje. Las células musculares lisas sembradas en forma de anillo total de agarosa pozos y el contrato de forma sólida en tres dimensiones (3D) de los tejidos dentro de 7 días. A escala milimétrica los anillos de tejido son propicias para los ensayos mecánicos y servir de base para el montaje del tejido.

Video: Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico andamios en ingeniería de tejidos arteriales

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Investigación

Bioingeniería

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo eléctrico controlado por migración dirigida de células progenitoras neurales en entornos 2D y 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Capillary Force Lithography for Cardiac Tissue Engineering

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

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

La evaluación de la administración de genes nanopartículas poliméricas por nanopartículas de análisis de seguimiento y de alto rendimiento Citometría de Flujo

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingeniería de tejidos de un humano 3D<em&gt; In vitro</em&gt; Sistema de Prueba de Tumores

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microentornos-Sandwich como para aprovechar Celulares / Materiales Interacciones

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Síntesis de cationizada Magnetoferritin para la magnetización ultra-rápido de las células

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Methods Article

Dirigida celular de auto-ensamblaje para fabricar células derivadas de tejido para el análisis de anillos de Biomecánica e Ingeniería de Tejidos