Nos gustaría dar las gracias a las granjas de Smithfield de la donación del tejido pulmonar porcino intacta. También nos gustaría agradecer al Dr. Hu Yang, el Dr. Christina Tang y el Departamento de Cirugía Plástica de VCU para permitirnos usar sus equipos. muestras de hidrogel y tejidos se prepararon para la SEM en el Departamento de Anatomía y de las instalaciones Neurobiología Microscopía VCU apoyado, en parte, por fondos del NIH NINDS Core Center de Grant 5 P30 NS047463 y, en parte, por la forma de financiación de los NIH-NCI Cancer Center Proyectos financiados P30 CA016059. SEM de imágenes de muestras en la Instalación Core Caracterización VCU Nanotecnología (NCC). Este trabajo fue financiado por la National Science Foundation, CMMI 1.351.162.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Solución </td><td> filtro estéril </td><td> Direcciones </td></tr><tr><td> DiH 2 O </td><td> Sí </td><td> DiH 2 O; estéril filtrada </td></tr><tr><td> Solución Triton X-100 0,1% </td><td> Sí </td><td> Bajo campana de humos Añadir 100 ml de Triton X-100 a 100 ml Solución DiH 2 O y agitar hasta que se disuelva; filtro estéril. </td></tr><tr><td> 2% desoxicolato Solution </td><td> Sí </td><td> Bajo campana de humos agregar 2 g de solución de desoxicolato de sodio por 100 ml de DiH 2 O y agitar hasta que se disuelva; filtro estéril </td></tr><tr><td> 1 M NaCl </td><td> Sí </td><td> Añadir 58.44 g de NaCl a 1 L de DiH 2 O; agitar hasta que se disuelva; filtro estéril </td></tr><tr><td> solución de DNasa </td><td> Sí </td><td> Añadir 12.000unidades de DNasa a 1 L DiH 2 O; Añadir 0,156 g MgSO4 (Anhidro), y 0,222 g de CaCl 2; agitar hasta que se disuelva; filtro estéril </td></tr><tr><td> PBS </td><td> Sí </td><td> Combinar 27,2 g de Na 2 HPO 4 · 7H 2 O (dibásico heptahidratado), 80 g de NaCl, y 2 g de KCl con 10 L DiH 2 O; agitar hasta que se disuelva; ajustar el pH a 7,4; filtro estéril </td></tr></tbody></table> Tabla 1: Soluciones requeridos para la descelularización de tejidos. Hacer las soluciones anteriores para el proceso de descelularización. Almacenar a 4 ° C. Aproximadamente se necesitará 2,5 L de un pulmón porcino. 1. Formación de hidrogel <ol><li> Porcino de pulmón descelularización (adaptado de referencias 9,10) <ol><li> Obtener, un cuarto bloque de pulmón porcino normal de un matadero, con el corazón y Vasculature intacto. </li><li> Con unas tijeras de tejido o un bisturí para diseccionar el corazón, vasculatura lo más cerca posible al corazón de corte, vasculatura restante se utilizó en pasos posteriores para perfusión. </li><li> diseccionar cuidadosamente el tejido conectivo que rodea la tráquea, los bronquios, y la vasculatura utilizando un escalpelo o tijeras. NOTA: Determinar el mejor de pulmón a utilizar para el resto del procedimiento por la elección de un pulmón y sin grandes heridas o perforaciones a través de la pleura y grandes cantidades de atelectasia o la oclusión vascular que pueden impedir la eficacia del proceso de descelularización. </li><li> Cortar el pulmón subóptima deja la mayor parte del bronquio unidos a la tráquea como sea posible (corazón, tejido conectivo y los lóbulos del pulmón eliminado podrán ser eliminados). Debe haber un pulmón que permanezcan unidas a la tráquea y la vasculatura. NOTA: Conservar secciones para histología si se desea. 9 </li><li> Cierre bronquios desconectados con abrazaderas o sutura a prevenir el exceso de flujo de retorno. </li></ol><ol start="6"><li> Preparar las soluciones descelularización y refrigerar a 4 ° C hasta que se necesite (Tabla 1). </li><li> El uso de una bomba de mano canulado para ajustarse a la arteria pulmonar, la perfusión del tejido pulmonar 3 veces con agua DI a través tanto de la arteria pulmonar y la tráquea. Perfundir vasculatura primera cada vez. Comienza con alrededor de 1 L en la vasculatura y 1,5 L en la tráquea para cada perfusión, pero como se elimina los desechos celulares más líquido puede ser perfundido a través del sistema. </li><li> Perfundir tanto vasculatura y la tráquea con una solución de Triton X-100. </li><li> Sumergir el tejido pulmonar en una solución de Triton X-100 durante 24 horas a 4 ° C. </li><li> Perfuse vasculatura y tráquea 3 veces con agua DI para enjuagar. </li><li> Perfundir tanto vasculatura y la tráquea con una solución de desoxicolato. </li><li> Sumergir las secciones de tejido en desoxicolato durante 24 horas a 4 ° C. </li><li> Perfuse vasculatura y tráquea 3 veces con agua DI para enjuagar. </li><li>Perfundir tanto vasculatura y la tráquea con una solución de NaCl. </li><li> Sumergir el tejido en solución de NaCl se filtró durante 1 hora a 4 ° C. </li><li> Perfuse vasculatura y tráquea 3 veces con agua DI para enjuagar. </li><li> Perfundir tanto vasculatura y la tráquea con una solución de DNasa. </li><li> Sumergir el tejido en solución filtrada DNasa durante 1 hora a 4 ° C. </li><li> Perfundir tanto vasculatura y la tráquea 5 veces con PBS. </li><li> Diseccionar tejido cartilaginoso notable, tráquea y todo túbulos 2 mm o más de diámetro (que se encuentra principalmente en torno a la hilio y porciones mediales de los pulmones) de la realización de las vías respiratorias, dejando sólo las zonas respiratorias (principalmente las zonas periféricas). </li><li> Diseccionar el tejido en secciones de 1 pulgada o más pequeños. Orientación del tejido no importa para este paso. </li><li> Retirar el exceso de tejido líquido y colocar en tubos de 50 ml cónicos. Congelar el tejido a -80 ° C. NOTA: Conservar secciones de histología para asegurar la eliminación de las células y debri celulars, si se desea. Utilizamos H &amp; E, α-galactosidasa, PicoGreen, hidroxiprolina, ninhidrina, Alcian azul, SDS-PAGE y espectrometría de masas para caracterizar. 9 </li></ol></li><li> Procesamiento de pulmón <ol><li> Retire las tapas de los tubos congelados que contienen tejido pulmonar descelularizado. </li><li> Coloque papel de filtro sobre la abertura del tubo y asegurar con banda de goma. NOTA: El tubo y el contenido todavía se deben congelar cualquier otra manera ponga en -80 ° C hasta que se congele. </li><li> Liofilizar el tejido hasta que todo el exceso de líquido se ha ido, utilizando un secador de congelación de acuerdo con las instrucciones del fabricante. Almacenar a -80 ° C hasta el momento de molino. </li><li> Antes de comenzar añadir nitrógeno líquido para molino congelador para enfriar el aislamiento y los componentes internos de las condiciones de trabajo. </li><li> Retire la barra magnética molino de tubo de molino y agregar el tejido para cubrir el fondo. </li><li> Cambie la barra de molino y agregar el tejido sin apretar. La barra de molino todavía debe moverse libremente. </li><li> Cerrar el tubo molino y place en el molino congelador. </li><li> Llenar nitrógeno líquido para línea de llenado máximo. </li><li> Molino Congelador todo el tejido en polvo fino (aproximadamente 5 min, la tasa de impactación de ~ 600 min -1), en un cilindro de policarbonato con un impactador de acero inoxidable, así como tapones de extremo de acero inoxidable utilizando el molino congelador de acuerdo con las instrucciones del fabricante. Almacenar a -80 ° C hasta que esté listo para su uso. </li></ol></li><li> Micro-porosa formación de gel (8 mg / ml) (adaptado de referencias 9,11) <ol><li> Añadir 1% (w / v) del polvo descelularizado y 0,1% (w / v) de pepsina a M HCl 0,01, con agitación constante (debe ser capaz de ver el flujo en el nivel superior de líquido), a temperatura ambiente, durante 48 hr. NOTA: El polvo se carga estáticamente, por lo que añadir el HCl después el polvo produce la oportunidad para lavar el exceso de las paredes del tubo. </li><li> Después de la digestión durante 48 h, colocar la solución y los reactivos en hielo durante 5 min. </li><li> Usando refrigerado 10% (v / v) de NaOH 0,1 M, unad 11.11% (v / v) 10x PBS (para llevar la solución entera a 1x concentración), llevar la solución de proteína digerida a pH fisiológico de 7,4. NOTA: La solución ahora se puede almacenar a 4 ° C durante un máximo de una semana. Realizar una cinética de gelificación utilizando reometría 9 si se desea. </li></ol></li></ol> 2. El entrecruzamiento hidrogeles para mejorar la resistencia mecánica <ol><li> Las soluciones de 1%, 0,1% y 0,01% w / v solución genipina reticulación se prepararon disolviendo la cantidad necesaria de polvo de genipina en 10% de DMSO. </li><li> Vortex la solución cada 15 min durante 1 hora. </li><li> Añadir solución genipina para cubrir hidrogeles ECM (100 l por cada pocillo de una placa de 96 pocillos) que han sido previamente montados en parte 1.3.4. </li><li> Dejar a cada hidrogel ECM para reticular durante 24 horas a 37 ° C. </li><li> Enjuague el hidrogel ECM 3 veces con PBS hasta que la solución de lavado ya no es de color azul. Los hidrogeles reticulados serán entonces listo para su posterior caracterización y ceestudios de cultivo ll. </li></ol> 3. cultivo celular con microporosa Gel <ol><li> Recubrimiento de dos dimensiones <ol><li> Usando la solución de 1.3.3, añadir 20 l de solución de pregel a cada pocillo de una placa de cultivo tisular de 96 pocillos no tratados. </li><li> refrigere durante la noche a 4 ° C para permitir la adsorción de proteínas a la placa. </li><li> Aspirar solución pregel y enjuague con PBS. </li><li> Células pasantes 9. </li><li> Añadir 10.000 células / cm 2 a los pocillos. </li><li> Aumentar los medios de comunicación a 100 l por pocillo y se incuba a 37 ° C. </li></ol></li><li> Tres de cultivo celular dimensional 9 <ol><li> Utilizando la solución de 1.3.3, resuspender las células sedimentadas a una concentración de 1.000.000 células / ml de solución pregel. </li><li> dispensar rápidamente 16 l de suspensión de células pregel en cada pocillo de una placa de 96 pocillos, para formar un hidrogel de espesor ~ 500 micras para el cultivo celular en 3D. </li><li> Incubar a 37 ° C durante 30min para el gel a la forma. </li><li> Añadir 100 l de medios de comunicación a la parte superior de hidrogeles formados y se incuba a 37 ° C. </li></ol></li><li> Tres de cultivo celular dimensional en geles de rigidez variable <ol><li> Utilizando la solución de 1.3.3, pipeta 100 l de solución de pregel en cada pocillo de una placa de 96 pocillos. </li><li> Incubar a 37 ° C durante 30 minutos para gel a la forma. Nota: El entrecruzamiento pasos de la parte 2 se puede utilizar aquí. </li><li> Células pasantes 9. </li><li> Añadir 10.000 células / cm 2 a los pocillos. </li><li> Aumentar los medios de comunicación a 100 l por pocillo y se incuba a 37 ° C. </li></ol></li></ol>

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E., Wu, J., Shah, M., Hong, Y., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Material+Properties+of+Cardiac+Matrix+Hydrogels+to+Induce+Endothelial+Differentiation+of+Human+Mesenchymal+Stem+Cells.">Tailoring Material Properties of Cardiac Matrix Hydrogels to Induce Endothelial Differentiation of Human Mesenchymal Stem Cells.</a> ACS Appl. Mater. Interfaces. 7 (20), 11053-11061 (2015).</li><li>Suckow, M. A., Voytik-Harbin, S. L., Terril, L. A., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bone+regeneration+using+porcine+small+intestinal+submucosa.">Enhanced bone regeneration using porcine small intestinal submucosa.</a> J. Invest. Surg. 12 (5), 277-287 (1998).</li><li>Brown, B. N., Badylak, S. 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R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS One. 5 (9), e12905 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+where+we+have+been+and+where+we+are+going.">Biomaterials: where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>Fridman, R., Benton, G., Aranoutova, I., Kleinman, H. K., Bonfil, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+initiation+and+growth+of+tumor+cell+lines,+cancer+stem+cells+and+biopsy+material+in+mice+using+basement+membrane+matrix+protein+(Cultrex+or+Matrigel)+co-injection.">Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.</a> Nat Protoc. 7 (6), 1138-1144 (2012).</li><li>Zhang, W. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reconstruction+of+lung+alveolus-like+structure+in+collagen-matrigel/microcapsules+scaffolds+in+vitro.">The reconstruction of lung alveolus-like structure in collagen-matrigel/microcapsules scaffolds in vitro.</a> J. Cell. Mol. Med. 15 (9), 1878-1886 (2011).</li><li>Booth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+Normal+and+Fibrotic+Human+Lung+Matrices+as+a+Culture+System+for+In+Vitro+Investigation.">Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation.</a> Am. J. Respir. Crit. Care Med. 186 (9), 866-876 (2016).</li><li>Matsuda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+human+lung+stem+cells.">Evidence for human lung stem cells.</a> N. Engl. J. Med. 364 (19), 1795-1806 (2011).</li><li>Zuo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p63+Krt5++distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63+Krt5+ distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Neill, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> Ann Thorac Surg. 96 (3), 1046-1055 (2013).</li></ol>

Los autores no tienen nada que revelar.

Uno de los aspectos integrales de la biología es la auto-organización de las moléculas en estructuras jerárquicas que realizan una tarea específica. 13 En el laboratorio, el autoensamblaje depende de numerosos factores tales como la concentración de sal, pH, y la duración de digestión. Como se muestra, se forma un hidrogel de auto-organización cuando las proteínas solubilizadas vuelve a la temperatura fisiológica. El hidrogel formado es capaz de promover la unión celular y la proliferación in...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Aquí presentamos un método para establecer múltiples hidrogeles de cultivo de células de componentes para el cultivo in vitro de células de pulmón en. Comenzando con sano en tejido bloque de pulmón de porcino, de rata, o un ratón, el tejido es perfundido y sumergido en detergentes químicos subsiguientes para eliminar los restos celulares. comparación histológica del tejido antes y después del procesamiento confirma la eliminación de más del 95% de la doble tinción de ADN y alfa galactosidasa hebra sugiere se elimina la mayoría de los restos celulares. Después de la descelularización, el tejido es liofilizada y después cryomilled en un polvo. El polvo de la matriz se digiere durante 48 horas en una solución de digestión con pepsina ácida y después se neutralizó para formar la solución pregel. La gelificación de la solución pregel puede ser inducida por la incubación a 37 ° C y puede ser utilizado inmediatamente después de la neutralización o se almacenaron a 4 ° C durante hasta dos semanas. Los recubrimientos se pueden formar usando la solución pregel en una placa no tratada para capego ell. Las células pueden ser suspendidas en el pregel antes de la auto-ensamblaje de lograr una cultura 3D, chapada en la superficie de un gel formado a partir de la cual las células pueden migrar a través del andamio, o chapada en los revestimientos. Las alteraciones en la estrategia presentada puede afectar la temperatura de gelificación, la fuerza o el tamaño de los fragmentos de proteínas. Más allá de la formación del hidrogel, la rigidez de hidrogel se puede incrementar utilizando genipina.

Usando este método, hemos producido hidrogeles de cerdo normal de rata, y los pulmones de ratón (Figura 1). pulmones procesados ​​proporcionan un estimado de 5 mg, 40 mg, y 10 g de polvo de ECM respectivamente. Una visión general del proceso se muestra en la Figura 2. visualizaciones clave durante el proceso incluyen: aspecto blanco de los pulmones después de aclarar desoxicolato; después de la formación pregel, la solución debe ser opaca y la...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Este es un método para crear un andamio de cultivo celular de 3 dimensiones de la matriz extracelular pulmonar. pulmonar intacta se procesa en hidrogeles que pueden apoyar el crecimiento de células en tres dimensiones.

Los hidrogeles sintonizable de la matriz extracelular pulmonar por la 3D de Cultivos Celulares

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Research

JoVE Journal

Bioengineering

11.5K vistas.  Virginia Commonwealth University.  Este es un método para crear un andamio de cultivo celular de 3 dimensiones de la matriz extracelular pulmonar. pulmonar intacta se procesa en hidrogeles que pueden apoyar el crecimiento de células en tres dimensiones. 

Video: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

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

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

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

Investigación

Bioingeniería

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

La fabricación de espumas y microportadores derivados de la matriz extracelular como específica de tejido de cultivo celular y las plataformas de entrega

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Hidrogeles ultrafino Porated elástico como una membrana del sótano biomiméticos para doble cultura de la célula

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabricación de un hidrogel de derivados de matriz extracelular renal corteza

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Producción de fibras de la matriz extracelular mediante cultivo de células de membrana de fibra hueca sacrificio

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Medición de metaloproteinasa de la matriz celular Global y actividad metabólica en hidrogeles 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioink de matriz extracelular derivada del tejido pancreático para imprimir construcciones de tejido pancreático laden de células 3D

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modelo de cultivo de matriz-adipocitos extracelulares 3D humano para el estudio de la conversación cruzada metabólica Matrix-Cell

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Preparación de microambientes de matriz extracelular sintonizable para evaluar la especificación del fenotipo celular Schwann

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Cepa controlada de hidrogeles 3D bajo imágenes de microscopía en vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...