Este trabajo fue apoyado por la Real Sociedad URF subvención UF051616, Reino Unido y el Consejo Europeo de Investigación StG subvención 243.261 BS. El trabajo en el laboratorio de MZ también es apoyado por un Instituto de California de Medicina Regenerativa de subvención RB1-01417.

1. Progenitoras neurales de aislamiento de células <ol><li> Diseccionar los cerebros enteros de E14-16 ratones y el lugar en frío DMEM/F12 medio basal. Quite todas las meninges con un microscopio anatómica y la transferencia de cerebros en un plato de Petri de 35 mm. </li><li> Use unas pinzas finas para disociar mecánicamente cerebros en fragmentos de tejido y los transfiere a un tubo de 15 ml, centrifugar las muestras a 800 rpm durante 3 minutos para eliminar los residuos. </li><li> Añadir DMEM/F12 que contiene bFGF y EGF y se tritura con una pipeta de 1 ml. </li><li> Pasar la suspensión celular a través de un filtro de células para obtener una suspensión de células individuales. </li><li> Células de la placa en frascos a 2-5 x 10 4 células / ml, a continuación, realizar un medio completo cambian cada 3 días y el paso de las células cada 6 días. </li><li> Después de al menos 5 pasajes, digerir neuroesferas a células individuales usando tripsina y EDTA y crecer en cámaras electrotactic poly-D-lysine/laminin-coated (preparado como se describe a continuación en 2). Utilice el medio de cultivo que contiene N2 suplemento,bFGF y EGF en todo momento para mantener las propiedades de los NPCs. </li></ol> 2. Preparación de la cámara de electrotactic <ol><li> Preparar 22 x 11 mm tiras de vidrio mediante la división en autoclave 22 x 22 mm de espesor cubreobjetos no.1 en medio utilizando una pluma de diamantes. </li><li> Crear un vaso independiente y de dimensiones interiores 22 x 10 mm al pegar cuatro verticalmente de pie 22 x 11 mm tiras con grasa de silicona de alto vacío. Permita que los pozos se sequen por completo y se endurecen durante la noche. </li><li> El día siguiente, conecte dos 22 x 11 mm tiras de vidrio, paralelos entre sí dejando un hueco de 10 mm, a la base de una placa de cultivo de 100 mm utilizando grasa de silicona. Sellar la región entre estas tiras mediante la colocación de un 22 x 22 mm cubreobjetos en cada extremo, unido a la parte inferior del plato con grasa en tres lados, pero no el más próximo al centro. </li><li> Coloque el cristal así preparada en la etapa 2,2 sobre la cubierta se desliza de modo que las paredes interiores crear un espacio confinado para la siembralas células en la parte inferior de la placa. A prueba de agua todas las juntas con grasa de silicona. Capa esta región confinada secuencialmente con poli-D-lisina entonces laminina: añadir 1 ml de poli-D-lisina en la cámara y se deja durante 5 minutos para permitir la poli-D-lisina de obligar a la parte inferior de la placa; lavar la cámara con PBS estéril dos veces, luego diluir laminina en PBS estéril para dar 20 mg / ml y utilizar para cubrir la parte inferior de la placa. Dejar a temperatura ambiente durante la noche. </li><li> El día siguiente, las células de la cosecha y preparar 1 ml de suspensión contiene de 1 x 104 células. Retire la laminina de la copa y, lo que permite que se seque por completo, y sustituir con 1 ml de suspensión celular. Poner la cápsula en la incubadora a 37 ° C durante un mínimo de 4 horas para permitir la fijación. </li><li> Una vez que las células son suficientemente confluente eliminar todo el medio de la cámara. Retire con cuidado el vaso bien. Forme un techo sobre las células por amarrando, con grasa de silicona, un autoclave de 22 x 22 mm cubreobjetospuente entre las dos tiras de 22 x 11 mm. Cubrir las células con unas pocas gotas de medio para evitar la desecación. </li><li> Formar un depósito medio de aislamiento en cada extremo de la cámara mediante la creación de dos barreras estancas de grasa de silicona que se ejecutan desde un borde del plato a la otra, sobre el techo de la cámara. Llenar la cámara con un nuevo medio, asegurar un flujo continuo desde el depósito medio una a la otra. Vuelva a colocar el plato a la incubadora durante 12 horas para permitir la recuperación celular. </li></ol> 3. La aplicación de un campo eléctrico a la cámara de electrotactic <ol><li> Preparar una tapa para cubrir el plato mediante la perforación de dos orificios, uno colocado sobre cada depósito de la cámara de la migración. </li><li> Sustituir todo el medio en la cámara con medio de cultivo que contiene 25 mM de tampón HEPES y transferir el plato para el sistema de imagen de temperatura controlada. Establecer los parámetros experimentales para time-lapse y grabación multi-posición. Alinear la cámara de manera que el cátodo y el ánodo sona la izquierda y derecha, respectivamente, para asegurar que el vector de EF se extiende horizontalmente como se ve por el microscopio y se registra en el sistema de imagen. </li><li> Llene dos vasos con una solución de Steinberg (58 mM de NaCl, 0,67 mM de KCl, 0,44 mM de Ca (NO3) 2 .4 H 2 0, 1,3 mM MgSO 4 .7 H 2 0 y 4,6 mm Trizma base, pH 7,4). Conectar un vaso de precipitados separado para cada depósito medio utilizando pre-preparadas puentes de vidrio (tubos de vidrio ~ 13 ~ cm de largo y 3 mm de diámetro, y doblado en forma de U por calentamiento en un mechero Bunsen), rellenas con 2% (w / v) Steinberg&#39;s-agar solución, que pasa a través de los orificios de la tapa. Complete el circuito eléctrico mediante la colocación de Ag / AgCl electrodos conectados a una fuente de alimentación de CC en cada vaso de precipitados de la solución de Steinberg. </li><li> Ajuste el dial de voltaje en el suministro de energía a 0 y se enciende. Medir la tensión a través de la cámara de electrotactic mientras se sube la tensión de línea, utilizando un medidor de voltaje, y ajustar para adaptarse a los requerimientos experimentales. </li><li> Inicio de lapso de tiempo de grabación. Realizar un cambio de medio y reajustar la tensión, según se requiera, cada hora. Medio fresco, drogas o agentes químicos se pueden añadir a los depósitos según sea necesario. Cuando realizar cambio de medio, dos opciones pueden ser considerados como a continuación: <ol><li> Opción N º 1 - Para poner en pausa la grabación de lapso de tiempo temporal, retire con cuidado los puentes de cristal de la cámara para evitar perturbar la tapa cubierta, utilice una pipeta Pasteur esterilizada vuelva a colocar todo el medio con un nuevo medio y colocar los puentes de cristal hacia atrás, a continuación, reanudar la grabación. </li><li> Opción N ° 2 - Alternativamente, hecho a medida tapa cubierta con 4 orificios (dos coloca sobre cada depósito de la cámara de la migración) también se puede utilizar para el propósito de cambio de medio. Dos de vidrio conexión puentes, los otros dos para cambiar medio. Opción 2 permite la grabación continua, sin interferencias durante el cambio de medio. </li></ol></li></ol> 4. Preparación de la rebanada de la médula espinal organotípico <ol><li> Diseccionar Lumbar la columna vertebral de los cables de 2 semanas de edad C57 BL / 6 ratones. </li><li> Cortar la médula espinal en 500 secciones micras de espesor con una picadora de tejidos McIlwain. </li><li> Separe las rebanadas en un microscopio anatómica y seleccionar sectores con intacta sagital / axial de la estructura de la médula espinal. </li><li> Rodajas de placa en una placa de Petri de 35 mm que contiene 30 l Matrigel y colocarlos lo más cerca posible del centro como sea posible y mantenerlos en un 5% de CO 2 incubadora a 37 ° C durante al menos 30 minutos hasta que el Matrigel proteínas se auto-ensamblan para producir una fina película que cubre la superficie de la rebanada de la médula espinal. Es muy importante asegurarse de que Matrigel ha sido montada por completo, se incuba la placa de Petri a 37 ° C durante otros 30 minutos si es necesario. </li><li> Añadir 4-6 ml DMEM/F-12 medio que contiene 25 mM de tampón HEPES y suero fetal de ternera 15-20% muy suavemente de modo que se evite el flujo medio directamente sobre la rebanada. Asegurarse de que la rebanada no está completamente sumergido en el medio, dejando la superficie de los explantes así expuestos ael aire. Cambiar medio dos veces por semana. </li></ol> 5. La inyección de Hoechst 33342 NPCs etiquetados en el segmento de la médula espinal organotípico <ol><li> Preparar la suspensión de la APN en el 1 × 10 6 células / l. </li><li> Preincubar la suspensión celular en un medio con 5 Hoechst 33342 mM durante 30 min. </li><li> Utilizar tubo capilar de vidrio a microinyectar 2 l de suspensión en el segmento de la médula espinal lentamente bajo un microscopio. Asegúrese tubo capilar de vidrio pasa a través del Matrigel (parte rosa bajo microscopio) y llegar al interior de la rebanada de la médula espinal (tejido gris bajo microscopio), siendo el interior de la rebanada de la médula espinal por lo menos 30 segundos para evitar la suspensión de células arrollado. Coloque la placa de Petri que contiene parte de la médula espinal en la incubadora (37 º C 5% 2) y se deja durante la noche. </li><li> Al día siguiente, aplicar una EF de 500 mV / mm para el corte de la médula espinal que contiene Hoechst 33342 que llevan la etiqueta de NPCs en la cámara de electrotactic (usando los métodos descritos en3). </li></ol> 6. Los resultados representativos Cuando NPCs fueron expuestos a una amplia gama de EF fisiológicos que mostraban la migración de células altamente dirigido hacia el cátodo (Figura 1). La misma observación se hizo también en un nivel de células individuales en el modelo de la columna vertebral organotípico rebanada de médula ex vivo, un entorno 3D que imitan las condiciones in vivo (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> Figura 1. PNJ muestran la migración dirigida de la SSC. PC, mostró una migración altamente dirigido hacia el cátodo cuando se expone a EF, líneas rojas y las flechas azules representan las trayectorias y la dirección de movimiento de las células (A). B muestra las rutas de migración de los NPCs. Bar: 50 m. <img alt="Figura 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> Figura 2. NPC trasplantados muestran migración dirigida hacia el cátodo en la rebanada de la médula espinal organotípico </strong&gt;. (A) NPC marcadas con Hoechst 33342 se trasplantaron en la rebanada de la médula espinal organotípico en el punto inicial del tratamiento de EF. NPC migrado direccionalmente hacia el cátodo durante 2,5 horas, momento en el que la polaridad se invirtió EF (B). La alteración de la polaridad EF provocó un drástico cambio de electrotaxis hacia el cátodo nueva (C). (D) Imagen de NPCs trasplantados dentro de la rebanada de la médula espinal en la final de la grabación de lapso de tiempo. (E) Una reconstrucción en 3D de los NPCs trasplantados en el segmento de la médula espinal. 3D secciones de exploración fueron 300 micras de espesor, a partir de la media y que termina en la parte inferior de la rebanada. Las líneas discontinuas indican las posiciones relativas de la misma población de células trasplantadas en el comienzo, la inversión, y los puntos finales del tratamiento FE (A - C, respectivamente). Puntas de flecha indican la misma población de Hoechst 33342 NPCs etiquetados. Bar: 50 m.

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

No tenemos nada que revelar.

Los protocolos que utilizamos se basa en estudios previos 5,11. El uso de estos métodos, la cultura estable y eléctrica actuales condiciones se puede mantener mientras se aplica una EF a través de puentes de agar, la solución de Steinberg, y los electrodos de Ag / AgCl, a las células o rodajas cultivadas en cámaras electrotactic diseño personalizado de dimensiones normalizadas y precisa. La profundidad de las cámaras puede ser ajustada para adaptarse a diferentes espesores de la muestra 11,...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> FGF-básico humano recombinante </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / ml </td></tr><tr><td> EGF humano recombinante </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / ml </td></tr><tr><td> N2-Suplemento (100X) de líquido </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> Medio DMEM/F12 (glucosa alta) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poli-D-lisina </td><td> Millipore </td><td> Una-003-E </td><td></td></tr><tr><td> Natural de laminina de ratón </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Factor de reducción del crecimiento de la membrana basal de matriz (Matrigel) </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> Tampón HEPES </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> McIlwain helicóptero del tejido </td><td> El Mickle Laboratorio de Ingeniería Co Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> Dow Corning de alto vacío grasa de silicona </td><td> Sigma-Aldrich </td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

Endógenos campos eléctricos (EFS) de forma natural en vivo y desempeñan un papel fundamental durante el desarrollo del tejido / órgano y la regeneración, incluida la de los 1,2 del sistema nervioso central. Estos FAN endógenos son generados por la regulación celular de transporte iónico en combinación con la resistencia eléctrica de las células y tejidos. Se ha informado de que aplicada EF tratamiento puede promover la reparación funcional de lesiones de la médula espinal en animales y humanos 3,4. En particular, EF-dirigida migración celular se ha demostrado en una amplia variedad de tipos celulares 5,6, incluyendo las células progenitoras neurales (NPC) 7,8. Aplicación de corriente continua (DC) FAN no es una técnica disponible en la mayoría de los laboratorios. Hemos descrito los protocolos de aplicación de los FAN DC a cultivos de células y tejidos previamente 5,11. Aquí presentamos un video de demostración de los métodos estándar basado en una intensidad de campo calculada para crear un 2Dd entornos 3D para los NPCs, y para investigar las respuestas celulares a la estimulación de EF en ambas condiciones de crecimiento de células individuales en 2D, y la porción de la médula espinal organotípico en 3D. El cordslice espinal es un tejido receptor ideal para el estudio del NPC comportamientos ex vivo, post-trasplante, debido a que la organización del tejido citoarquitectura está bien conservada dentro de estas culturas 9,10. Además, este modelo ex vivo también permite que los procedimientos que no sean técnicamente viables para rastrear las células in vivo utilizando lapso de tiempo de grabación en el nivel de células individuales. Es sumamente esencial para evaluar el comportamiento celular no sólo en un entorno 2D, sino también en una condición 3D organotípico que imita el entorno in vivo. Este sistema permitirá a imágenes de alta resolución con cubierta a base de platos de cristal en el tejido o el cultivo de órganos con seguimiento 3D de la migración celular solo in vitro y ex vivo y puede ser un paso intermedio antes de pasar a en vivo paradigmas.

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

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

Este protocolo muestra los métodos utilizados para establecer entornos 2D y 3D en las cámaras de electrotactic de diseño personalizado, que puede rastrear las células<em&gt; In vivo / ex vivo</em&gt; Con lapso de tiempo de grabación en el nivel de células individuales, a fin de investigar galvanotaxia o electrotaxis y otras respuestas celulares a la corriente directa (DC) los campos eléctricos (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 ...

electric-field-controlled-directed-migration-neural-progenitor-cells

Research

JoVE Journal

Bioengineering

11.6K vistas.  Cardiff University. Este protocolo muestra los métodos utilizados para establecer entornos 2D y 3D en las cámaras de electrotactic de diseño personalizado, que puede rastrear las células In vivo / ex vivo Con lapso de tiempo de grabación en el nivel de células individuales, a fin de investigar galvanotaxia o electrotaxis y otras respuestas celulares a la corriente directa (DC) los campos eléctricos (EFS).

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

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

Adaptación de un Robot Haptic en un fMRI 3T

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Investigación

Bioingeniería

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de Células progenitoras neurales en un hidrogel de péptidos 3-dimensional de auto-montaje

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Ordenando las células individuales y solo los embriones en 3D Confinamiento: un nuevo dispositivo para la selección de alto contenido

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Modelo de polarizado Neural tisular basada Silk-colágeno-Engineered 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Estudios electrotaxis de pulmón células de cáncer utilizando un Multicanal Dual-campo eléctrico chip de microfluidos

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

La mejora en 2D y 3D de la piel<em&gt; In Vitro</em&gt; Uso de modelos macromoleculares hacinamiento

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Matrices 2D y 3D para estudiar la formación y la actividad invadosoma lineal

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Caracterización de la migración de la célula en tres dimensiones In Vitro herida ambientes

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Mejora 3D hidrogel culturas de células Glial primarias para In Vitro modelado de neuroinflamación

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

Manipulación de células madre y neuronas neuronales únicas en las rebanadas cerebrales mediante la microinyección robótica

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...