El financiamiento fue proporcionado por los Institutos Nacionales de Salud (EB 001 013), Ciencias Naturales e Ingeniería de Investigación de Canadá y los Institutos Canadienses de Investigación en Salud. Agradecemos al Dr. AP McGuigan por su experiencia y ayudar en el desarrollo de los módulos.

1) Preparación del tubo <ol><li> Cortar 2 a 3 m de longitud de tubo de polietileno (0,76 mm de diámetro x 1,22 mm OD). </li><li> Enhebrar una aguja 20 G1 en un extremo de la tubería. </li><li> Bobina de la tubería y el lugar en un convertidor de autocierre bolsa (7 1 / 2 &quot;x 13&quot;). Los dos extremos de la tubería debe ser colocado en el extremo sin sellar de la bolsa y se pega con cinta adhesiva de gas de esterilización. </li><li> Selle la bolsa y el gas esterilizar. </li></ol> 2) La neutralización de colágeno Nota: 1 ml de colágeno se llena de unos 2 m de tubería de polietileno. <ol><li> Coloque la cantidad necesaria de colágeno necesario para la longitud deseada del tubo en un tubo cónico de 15 ml y mantener en hielo. </li><li> Añadir 128 uL de 10x α-MEM por ml de colágeno en el tubo y mezclar varias veces la solución de pipeta. Nota: (1) La solución debe a su vez el amarillo como el α-MEM se mezcla con el colágeno acidificado y (2) tratar de minimizar las burbujas de la mezcla. </li><li> Neutralizar el colágeno mediante la adición de bicarbonato de sodio 0,8 M hasta que el colágeno tiene un pH de 7,4. Para una estimación general de que el bicarbonato de sodio pH añadir hasta que la solución de colágeno amarilla se vuelve de un color rosa claro. Nota: Por lo general toma 30 a 60 l por cada ml de colágeno. </li><li> Mantener el colágeno neutralizado en el hielo hasta que esté listo para su uso como el colágeno se neutraliza si no gel se mantiene a 4 ° C. </li></ol> 3) Incorporación de las células (Opcional) Nota: Dependiendo del tipo de células, las células pueden ser incorporados a una concentración de entre 1 millón de células por ml a 20 millones de células por ml. Utilizar el medio necesario para las células que desea incrustar. <ol><li> Eliminar los medios de comunicación de las células y lavar con 5 ml de PBS. </li><li> Quitar PBS y añadir 3 ml de tripsina-EDTA. </li><li> Se incuban las células en tripsina-EDTA durante 5 min a 37 ° C. </li><li> Resuspender las células en 7 ml de medio. </li><li> Recuento de células. </li><li> La transferencia de la cantidad necesaria de células para incorporar a un tubo cónico de 15 ml. </li><li> Que sedimenten las células a 300 xg durante 5 min. </li><li> Aspirar los medios de comunicación de la pellet de células. </li><li> Resuspender las células en el colágeno neutralizado y mantener en hielo. </li></ol> 4) gelificantes de colágeno <ol><li> En una cabina de seguridad biológica cortar el extremo cerrado de la bolsa que contiene el tubo de polietileno. </li><li> Adjunte una jeringa de 3 ml a la aguja 20G en el extremo del tubo con unas pinzas esterilizadas. Mantener el tubo en la bolsa. </li><li> Inserte el otro extremo de la tubería en el colágeno neutralizado hasta que la punta está en la parte inferior del tubo cónico con unas pinzas esterilizadas. Mantenga el tubo cónico en el hielo. </li><li> Tire del colágeno en la tubería de polietileno de retraer el émbolo de la jeringa lentamente. Nota: Si el colágeno se tira en el tubo de forma rápida luego las burbujas se forman en el colágeno. </li><li> Después de tirar el colágeno a través del tubo, saque la aguja 20G de la tubería y colocar los dos extremos en la bolsa. Cinta de la bolsa cerrada. </li><li> Incubar la bolsa a 37 ° C durante 60 min. Si el colágeno contiene células después poner la bolsa sobre cada 15 minutos. de modo que las células no se depositan en un lado del módulo. </li></ol> 5) El corte de tubo que contiene el colágeno gelificado <ol><li> Encabezados autoclave y la hoja de la herramienta de corte y bandeja para tubos. </li><li> Montar el dispositivo de corte. </li><li> Tubo lugar en una bandeja estéril frente de herramienta de corte y carga del tubo en el dispositivo de corte. </li><li> Establecer un tubo cónico de 50 ml que contienen 25 ml de los medios de comunicación a la salida del dispositivo de corte para recoger los módulos de corte. Nota: Use los medios que contiene FBS, incluso para el colágeno de sólo los módulos que los módulos no se separan de la tubería sin FBS en los medios de comunicación. Si los módulos contienen las células utilizan los medios necesarios para que las células incrustado. Si no hay células incluidas en los módulos de utilizar los medios de comunicación de las células endoteliales. </li><li> Configurar el dispositivo para el corte 2 mm de longitud de la tubería y cortar el tubo. </li><li> Incubar corte módulos a 37 ° C durante 60 minutos en el tubo cónico de 50 ml. </li></ol> 6) Extracción de módulos de colágeno de la tubería <ol><li> Vortex 50 ml tubo cónico que contiene los módulos en el tubo durante 10 segundos. Nota: Tenga cuidado al separar los módulos integrados de alta densidad celular para evitar roturas. </li><li> Deje que los módulos se depositan en el fondo del tubo. Se tarda unos 5 minutos. </li><li> Usando una pipeta de boca ancha 10 ml serológicas de transferencia de los módulos se establecieron en un tubo cónico de 15 ml. </li><li> Repita los pasos 6,1 hasta 6,3 veces. </li><li> Vaya al paso 7 de módulos capa con las células endoteliales o módulos de transferencia de una placa de cultivo de tejidos no tratados. </li></ol> 7) Revestimiento de los módulos con las células endoteliales <ol><li> Resolver los módulos en la parte inferior de un tubo cónico de 15 ml con 5 ml de los medios de comunicación para THe integrado células. </li><li> Retire el medio de las células endoteliales y lavar con 5 ml de PBS. </li><li> Quitar PBS y añadir 3 ml de tripsina-EDTA. </li><li> Se incuban las células en tripsina-EDTA durante 5 min a 37 ° C. </li><li> Resuspender las células en 7 ml de medio. </li><li> Recuento de células. Necesidad de 5x10 6 células endoteliales por ml de módulos de lleno en la parte inferior del tubo cónico. </li><li> Que sedimenten las células a 300 xg durante 5 minutos y resuspender en 5 ml de medio de las células endoteliales. </li><li> Agregar los 5 ml de los medios de comunicación que contiene las células endoteliales de los módulos. Nota: Esto le da una proporción de 50:50 de los dos tipos de medios que hemos encontrado para trabajar para la supervivencia y la función de ambos tipos celulares. Probar el uso de un medio de co-cultivo formulado para garantizar un mantenimiento adecuado de los fenotipos de los dos tipos de células antes de usarlo. </li><li> Incubar los módulos con las células endoteliales tanto estática como dinámica. </li><li> Para los módulos de incubación estática se mezclan con las células endoteliales de la inversión y colocar el tubo en posición vertical a 37 ° C durante 15 min. Repetir. </li><li> Dinámica de semillas de las células endoteliales agitando suavemente el tubo a 37 ° C durante 60 min. </li><li> Repita dos veces incubación estática. </li><li> Coloque los módulos en una placa de cultivo de tejidos no tratados y se incuba durante la noche a 37 ° C. </li><li> El lugar al día siguiente de los módulos en una placa de cultivo de nuevos tejidos no tratados con medio fresco (mezcla 50:50 si se utiliza el sistema de co-cultivo) para eliminar las células endoteliales sin ataduras. </li><li> Incubar los módulos que están cubiertas con células endoteliales hasta cubrir el 100% de la superficie de los módulos. Esto generalmente toma alrededor de 7 días. </li></ol> 8) Los resultados representativos: Los módulos se verá cilíndrica la primera vez que se retiran del tubo. Si los módulos contienen células integradas y / o están cubiertas con las células endoteliales que se pueden contratar hasta un 50% en volumen y desarrollan una forma oval (Figura 1). Los módulos también se volverá más densa y más opaco cuando es visualizado mediante microscopio de luz (Figura 1). Además, cuando las células endoteliales son confluentes en la superficie de los módulos hay una formación de uniones estrechas que se puede ver por inmunofluorescencia de VE-cadherina (Figura 2). El análisis de transferencia de masa se ​​ha demostrado que los módulos son capaces de soportar altas densidades de células (8x10 7 células / cm 3), sin el desarrollo de un núcleo necrótico debido al transporte inadecuado de oxígeno, que es a menudo problemático en las grandes tejidos (por ejemplo, módulos de gran diámetro, D = 1,4 mm) (Figura 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Figura 1" /> Figura 1: Módulo de fabricación y la contracción. Contracción del módulo durante los tres días siguientes a HUVEC-C (línea de células endoteliales) siembra resultados significativamente menor diámetro y la longitud del módulo (p &lt;0,001) (A). Incorporado las células HepG2 fueron distribuidos de manera uniforme dentro de los módulos en el momento de la fabricación y mantiene una alta viabilidad (B). [En vivo las células verdes, las células muertas de red] [adaptado de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Figura 2" /> Figura 2: VE-cadherina tinción de inmunofluorescencia de las uniones celulares CE apretado 10 días después de RAEC en cubierta en la superficie de un módulo. Barra de escala = 50 micras. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Figura 3" /> Figura 3: tricrómico de Masson de módulos típicos (0,76 mm de diámetro inicial) y los módulos de gran tamaño (1,4 mm de diámetro inicial) demostrar el efecto de las limitaciones de la difusión de oxígeno. Siete días después de la fabricación, un gran número de células muertas se había formado en el núcleo de los módulos de gran tamaño (panel inferior derecho), dejando sólo un delgado borde (~ 200μm de espesor) de células viables. Por el contrario, los pequeños módulos mantenido una distribución uniforme y elevado de células vivas. [Módulos integrados con las células HepG2 y recubierto con las células endoteliales.] [Tomado de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Figura 4" /> Figura 4: (A) HMEC-1 cabeza de serie en poloxamina - módulos de colágeno. Después de un día de la siembra de los módulos de colágeno poloxamina-mantienen su forma cilíndrica y no se limita propiedades adhesión celular en comparación con los módulos de colágeno solamente. Barra de escala = 200 micras. (B) Luz imagen microscópica de un módulo de colágeno que contiene PLGA basada en microesferas biodegradables. Barra de escala = 500 micras. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Figura 5" /> Figura 5: Ejemplos de ensayos in vitro. (A) ensayo de la angiogénesis: capilar como formaciones en el módulo de sembrado con las células endoteliales puede ser fácilmente detectado y cuantificado después de 5 días de incubación con las células madre derivadas de tejido adiposo. (B) las imágenes de microscopía confocal de un ensayo en vivo / muerto en los módulos donde la sTaining, de células vivas es de color verde y de células muertas es de color rojo. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Figura 6" /> Figura 6: Módulos de colágeno que contiene incrustado células madre mesenquimales y una capa superficial de células endoteliales. Los módulos fueron expuestos a la tensión de corte (~ 0,64 dinas / cm 2) durante 7 días en una cámara de microfluidos y comenzar a mostrar la fusión en los puntos de contacto. Las células endoteliales son teñidas con vWF (marrón) y los núcleos de células madre mesenquimales aparecen de color azul (H &amp; E). Barra de escala = 100μm. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Figura 7" /> Figura 7: Cientos de módulos que contienen colágeno derivadas de tejido adiposo-Las células madre (ASC) y recubierto con células endoteliales microvasculares (HMEC) se implantan bajo la piel de ratones para estudiar ASC / HMEC interacción in vivo para las aplicaciones de la regeneración de la grasa.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

Hemos fabricado varias diferentes micro-tejidos con módulos integrados con diferentes tipos de células 1.11. Hemos incorporado con éxito los cardiomiocitos primarios, islotes, las células adiposas madre mesenquimales y las células del estroma, así como varias líneas celulares HepG2 incluyendo, NIH 3T3 y clonar las células del hígado 9. Hemos revestido los módulos con varios tipos de células incluyendo las células endoteliales de aorta de rata endoteliales, las células endoteliales de vena umbilic...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
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fabrication of micro-tissues using modules of collagen gel containing cells

Este protocolo describe la fabricación de un tipo de micro-tejidos llamados módulos. El concepto de módulo genera tejidos uniforme, escalable y vascularizado. Los módulos pueden ser hechos de colágeno, así como otros materiales gelable o reticulables. Son aproximadamente 2 mm de longitud y 0,7 mm de diámetro en la fabricación, pero disminuye de tamaño con las células incrustado o cuando los módulos están cubiertos con las células endoteliales. Los módulos individuales son tan pequeñas que las células están incrustadas dentro del límite de la difusión de oxígeno y otros nutrientes, pero los módulos se pueden agruparse para formar los tejidos que son más grandes perfusable. Estos tejidos son modulares en la construcción debido a los diferentes tipos de células pueden ser integrados en cubierta o en los módulos antes de que se empaquetan para formar tejidos complejos. Existen tres pasos principales para hacer los módulos de: (1) neutralizar el colágeno y la incrustación de células en el mismo, (2) gelificantes el colágeno en el tubo y corte de los módulos y (3) revestimiento de los módulos con las células endoteliales.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

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

Creación de micro-tejidos, utilizando geles de colágeno cilíndricos, llamados módulos, que contienen células incluidas y que la superficie está recubierta de células endoteliales.

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

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

fabrication-micro-tissues-using-modules-collagen-gel-containing

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JoVE Journal

Bioengineering

12.6K vistas.  University of Toronto. Creación de micro-tejidos, utilizando geles de colágeno cilíndricos, llamados módulos, que contienen células incluidas y que la superficie está recubierta de células endoteliales.

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

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabricación de hidrogeles micropatterned para sistemas de cultivo neuronal usando fotolitografía dinámica de proyección Máscara

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Investigación

Bioingeniería

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging desnaturalizadas colágeno Strands<em&gt; En vivo</em&gt; Y<em&gt; Ex vivo</em&gt; A través de Photo-desencadenada Hibridación de Caged colágeno Mimetic Péptidos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

El cartílago humano Tissue Fabrication Usando tridimensional Tecnología de impresión inyección de tinta

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tejido picado en colágeno comprimido: Un Bio Transplant que contiene células para la reparación reconstructiva-puesta en escena individual

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Análisis preparación y fotoacústica de Vehículos celulares que contienen oro Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formando polímerosomas de tamaño gigante Uso de rehidratación asistida-Gel

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricación y caracterización de tejido óptico fantasmas que contiene macroestructura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparación, purificación y uso de liposomas que contienen ácidos grasos

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Absorción de nuevas nanopartículas recubiertas de lípidos que contienen Falcarindiol por células madre mesenquimales humanas

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Producción microfluídica de liposomas sensibles a la temperatura que contienen lysolipido

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...