Los autores desean agradecer a Baden-Württemberg Stiftung por su apoyo financiero en el marco de la "Síntesis de materiales Bioinspirados" (biomantos-14).

1. hidrogel preparación
<ol>
	<li>Mezclar 200 mg de BSA con 1 mL de desionizada de H2O para crear acciones de BSA 20% (p/v) (solución stock A).</li>
	<li>Mezcla 165 μl de solución de THPC (134 mg/mL) con 4,835 mL de agua desionizada para crear THPC stock (solución stock B).</li>
	<li>Pesar 1 mg de KCSSGKSRGDS (1,111.1 g/mol) péptido (o un péptido celular adhesiva equivalente) y diluirlo en 100 μl de estéril de H2O para obtener una solución de 10 mg/mL (solución C). Nota: Este paso es opcional y sólo debe incluirse si el hidrogel es para uso de cultivo celular.</li>
	<li>Quitar la parte inferior de una placa de 96 pocillos y reemplazarlo con envoltura de plástico extraíble. Nota: Para las placas de aquí, la parte inferior se puede quitar fácilmente aplicando presión a la parte inferior de cada pocillo; el fondo plástico delgado simplemente se cae.</li>
	<li>Mezclar 100 μl de solución stock de BSA (A) y 100 μl de THPC stock solución (B) (opcional: 4 μL de solución madre C para hidrogeles funcionalizados, adhesivo de célula) en la placa de 96 pocillos para obtener 200 μL de hidrogel. Mezclar los componentes mediante pipeteo arriba y abajo por lo menos 5 veces para garantizar un hidrogel uniforme después de la polimerización.</li>
	<li>Coloque la placa de 96 pocillos a temperatura ambiente (RT) durante unos 10 minutos hasta que los hidrogeles se polimerizan correctamente.</li>
	<li>Cuidadosamente y lentamente retire el envoltorio plástico de la parte inferior de la placa.</li>
	<li>Presione los hidrogeles de la placa de 96 pozos utilizando un sello pequeño y transferirlas a tubos de 1,5 mL con PBS estéril, pH 7,4. Nota: Los hidrogeles tienen una forma cilíndrica, con un diámetro de aproximadamente 4 mm y 8 mm de altura.</li>
	<li>Guarde los hidrogeles en solución salina tamponada con fosfato (PBS) a 4 ° C por hasta varios meses.</li>
</ol>
2. los hidrogeles de la liofilización
<ol>
	<li>Llenar tubos de 1,5 mL con 500 μl de estéril desionizada H2O y retire el tapón. Transferencia de los hidrogeles a los tubos de reacción de 1,5 mL con una espátula.</li>
	<li>Envuelva los tubos de 1,5 mL con parafina película-al menos tres capas de cada frasco. Utilice una aguja para perforar pequeños agujeros en la película para permitir la liberación de gas del tubo.</li>
	<li>Proceder a uno de los siguientes pasos:
	<ol>
		<li>Transferir el frasco de solución de nitrógeno líquido durante 5 minutos garantizar que el agua y el hidrogel congelan completamente. Inmediatamente después de la eliminación de nitrógeno líquido, transferir los frascos a la congelación del secador para evitar que el material descongelado. Nota: Este procedimiento resulta en poros de 10-15 μm de radio.</li>
		<li>Mantener los frascos a-20 ° C durante la noche para congelar lentamente los hidrogeles. Inmediatamente después del retiro de-20 ° C, transferir los frascos a la congelación del secador para evitar que el material descongelado. Nota: Este procedimiento resulta en poros de 50-60 μm de radio.</li>
	</ol>
	</li>
	<li>Después de 24 h y la completa evaporación del agua en y alrededor el hidrogel, descongelar el material mediante la eliminación de la congelación del secador. Nota: Los tamaños de poro pueden ser analizados con láser confocal de barrido microscopía (paso 5, "Visualización de hidrogel").</li>
</ol>
3. partículas de lixiviación
<ol>
	<li>Preparar el hidrogel como se describe en los pasos 1.1-1.5.</li>
	<li>Después de mezclar los componentes, agregar directamente NaCl hasta saturación (36 mg/mL). Añadir la sal hasta un cristal de sal puede ser visto en la solución como un precipitado blanco.</li>
	<li>Transferir la placa de 96 pocillos a una coctelera y agite hasta que la polimerización tiene lugar (aproximadamente 10 min). Quite los hidrogeles de la placa, como se describe en pasos 1.7-1.8.</li>
	<li>Incubar los hidrogeles por al menos 24 h a temperatura ambiente en agua estéril en un agitador (20 rpm) a fin de eluir toda la sal de la plantilla de hidrogel. Almacenar los hidrogeles a 4 ° C en PBS por hasta varios meses.</li>
</ol>
4. canal formación
<ol>
	<li>Preparación de hidrogeles como se describe en el paso 1. Quitar un hidrogel de solución utilizando una pinza, quitar el exceso de agua con papel muy absorbente y colocarlo encima de un bloque de hielo seco.</li>
	<li>Congelar el hidrogel durante 30 s y retire cuidadosamente el bloque. No dañe el hidrogel; cuidadosamente con una espátula raspe el bloque. El hidrogel de transferencia a un tubo de 1,5 mL y secar toda la noche a 37 ° C.</li>
</ol>
5. hidrogel visualización
<ol>
	<li>Preparar una solución stock de rodamina B diluyendo 1 mg de rodamina B en 10 mL de PBS. Preparar una dilución seriada diluyendo la solución madre de rodamina en PBS hasta alcanzar una concentración de 0.001 mg/mL (factor de dilución: 100)</li>
	<li>Retire el hidrogel de la solución de almacenamiento (por ejemplo, en el paso 2.4.) y transferirlo a un tubo de 1,5 mL con 1 mL de solución de rodamina B 0,01 mg/mL. Mancha el hidrogel durante la noche en solución de rodamina B a TA.</li>
	<li>Al día siguiente, transferir el hidrogel que debe visualizarse a 10 mL de PBS (pH 7.4) y lavado durante al menos 3 horas.</li>
	<li>Transferir el hidrogel en un μ-diapositiva 8 bien y cubrirla con PBS.</li>
	<li>Cortar rodajas pequeñas del hidrogel (sobre 0,5 mm de altura) con una hoja.</li>
	<li>Usando un láser confocal de barrido microscopio, visualizar el hidrogel en una longitud de onda de 514 nm (objetivo: CE Plan-Neofluar 40 x / 1.30 aceite DIC M27, modo de escaneo plano: 514 nm y zoom: 1 X).</li>
</ol>
6. célula cultura viabilidad
<ol>
	<li>Filtro estéril todo stock soluciones (A, B y C) con un filtro de 0.45 μm.</li>
	<li>Preparar el hidrogel como se describe en los pasos 1.1-1.4, incluyendo el péptido de la célula-adhesivo antes de la polimerización de hidrogel.</li>
	<li>Después de mezclar los componentes, pipetear inmediatamente la mezcla en una μ-diapositiva 8 bien hasta la parte inferior está totalmente cubierta. Nota: Este paso debe realizarse rápidamente y directamente después de mezclar los componentes, como la polimerización ocurre dentro de minutos en la diapositiva.</li>
	<li>Cultura adhesión células y paso para obtener una suspensión unicelular utilizando técnicas de cultivo celular estándar. Transferir las células a precalentado, estéril medio de cultivo celular DMEM suplementado con suero bovino fetal (FBS, 10% (p/v)), penicilina-estreptomicina (1% (p/v)) y la solución de aminoácido no esencial (MEM, 1% (p/v)).</li>
	<li>Contar las células con un Neubauer contando a cámara y cuidadosamente 200 μL de pipeta del número de células (2 x 105 células/cm2) en la superficie del hidrogel.</li>
	<li>La μ-diapositiva 8 con la tapa de la cubierta y transferirlo a una incubadora (37 ° C, 5% CO2). Incubar durante al menos 4 h a 37 ° C.</li>
	<li>Después de al menos 4 h de accesorio de celular, se lavan las células dos veces con 200 μL de PBS de la cultura de célula estéril.</li>
	<li>Fijar las células con 200 μL de formaldehído de 3.7% (v/v) durante 10 min a temperatura ambiente y lavar dos veces con PBS (~ 200 μL). Nota: Use equipo de protección personal cuando maneje formaldehído.</li>
	<li>Permeabilizar las células con 200 μL de 0,1% Triton X 5 min lavado dos veces con PBS.</li>
	<li>Se tiñen las células con phalloidin-rodamina mezclando 5 μl del stock metanólica con 195 μl de PBS y agregarlo a las células en RT. mancha las células durante 20 min en la oscuridad. Lavar dos veces con PBS.</li>
	<li>Investigar las propiedades de adherencia de células utilizando un microscopio confocal en 514 nm (objetivo: CE Plan-Neofluar 40 x / 1.30 aceite DIC M27, modo de escaneo plano: 514 nm y zoom: 1 X). Analizar las imágenes utilizando el software apropiado (véase la Tabla de materiales).</li>
</ol>
7. hidrogel propiedades
<ol>
	<li>Relación de la inflamación.
	<ol>
		<li>Secar completamente los hidrogeles a 37 ° C durante al menos un día.</li>
		<li>Pesar cada hidrogel y anote el peso exacto.</li>
		<li>Llene un tubo de ensayo de 2 mL con 1,5 mL de PBS.</li>
		<li>Transferir el hidrogel en este tubo de ensayo 2 mL y sumergirlo completamente en PBS.</li>
		<li>Deje el hidrogel durante al menos dos días en PBS a temperatura ambiente para alcanzar el equilibrio con el PBS.</li>
		<li>Después de tres días, retire el hidrogel de la solución y seque con un pañuelo de papel para eliminar el exceso de agua de la superficie del hidrogel.</li>
		<li>Peso del hidrogel.</li>
		<li>Calcular la relación hinchazón mediante la fórmula siguiente: 
		<img alt="Equation" src="/files/ftp_upload/55813/55813eq1.jpg"> donde Wd es el peso del gel seco (paso 7.1.2.) y Ws es el peso del gel húmedo (paso 7.1.7). Multiplicar con 100 para obtener el porcentaje de cociente de la hinchazón.</li>
	</ol>
	</li>
	<li>estabilidad de pH y temperatura.
	<ol>
		<li>Transferir 5 mL de PBS a un tubo de ensayo de 15 mL.</li>
		<li>Ajustar la solución para el pH apropiado (por ejemplo, pH 2, 7 o 10) con NaOH y HCl. traer la solución a la temperatura apropiada (p. ej., RT, 37 ° C y 80 ° C).</li>
		<li>Transferir el hidrogel que es investigada por su estabilidad en la solución adecuada. Vuelva a ajustar el pH si es necesario. Nota: Todos los hidrogeles deben ser completamente hinchados en este momento (ver la hinchazón) para evitar la interpretación errónea del peso debido a la hinchazón del material.</li>
		<li>Después de ciertos intervalos de tiempo, retire el hidrogel de la solución (por ejemplo, cada hora hasta 2 días), secar con un pañuelo de papel muy absorbente para quitar exceso de agua y pesar.</li>
	</ol>
	</li>
	<li>Degradación enzimática.
	<ol>
		<li>Preparar una solución stock de enzima de 300 U tripsina y pepsina, según las instrucciones del fabricante.</li>
		<li>Transferir el hidrogel (p. ej., de paso 1.8) a la solución adecuada. Nota: Todos los hidrogeles deben estar completamente inflamados en este punto para evitar la interpretación errónea del peso.</li>
		<li>Después de ciertos intervalos de tiempo, retire el hidrogel de la solución (por ejemplo, cada hora), secar con un pañuelo de papel muy absorbente para quitar exceso de agua y pesar.</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspheres+leaching+for+scaffold+porosity+control.">Microspheres leaching for scaffold porosity control.</a> J Mater Sci. 16 (12), 1093-1097 (2005).</li><li>Whang, K., 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=Engineering+Bone+Regeneration+with+Bioabsorbable+Scaffolds+with+Novel+Microarchitecture.">Engineering Bone Regeneration with Bioabsorbable Scaffolds with Novel Microarchitecture.</a> Tissue Eng. 5 (1), 35-51 (1999).</li><li>Ziv, K., 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=A+tunable+silk-alginate+hydrogel+scaffold+for+stem+cell+culture+and+transplantation.">A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation.</a> Biomaterials. 35 (12), 3736-3743 (2014).</li><li>Butruk-Raszeja, B. 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=Athrombogenic+hydrogel+coatings+for+medical+devices--Examination+of+biological+properties.">Athrombogenic hydrogel coatings for medical devices--Examination of biological properties.</a> Colloid Surface B. 130, 192-198 (2015).</li><li>L&#252;, S., Li, B., Ni, B., Sun, Z., Liu, M., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+injectable+hydrogel+for+three-dimensional+cell+culture:+chondroitin+sulfate+bioconjugated+with+poly(N-isopropylacrylamide)+synthesized+by+RAFT+polymerization.">Thermoresponsive injectable hydrogel for three-dimensional cell culture: chondroitin sulfate bioconjugated with poly(N-isopropylacrylamide) synthesized by RAFT polymerization.</a> Soft Matter. 7 (22), 10763 (2011).</li><li>Ruoslahti, E., Pierschbacher, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+in+cell+adhesion:+RGD+and+integrins.">New perspectives in cell adhesion: RGD and integrins.</a> Science. 238 (4826), 491-497 (1987).</li></ol>

Los autores declaran que no tienen intereses financieros que compiten.

The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of th...

Los hidrogeles son materiales que forman redes 3D insolubles capaces de atar grandes cantidades de agua. Este tipo de materiales puede proporcionar excelentes condiciones ambientales para las células vivas. Actualmente, existe un creciente interés en la generación de estructuras tridimensionales de hidrogel y en el desarrollo de los procesos para adaptar sus propiedades químicas y físicas. Una vez que esto se logra, una plantilla para el crecimiento de las células y la manipulación del comportamiento celular puede ser generado1,2,3,4. Estas estructuras 3D no sólo crean un ambiente más natural y realista que los enfoques convencionales de dos dimensiones, pero también revelan nuevas posibilidades para el crecimiento de las células madre o tumor modelos5. Diferentes materiales poseen una serie de características que dependen principalmente del tamaño del poro del gel6. Los poros juegan un papel crucial en aplicaciones de cultivo celular, ingeniería de tejidos y el crecimiento dirigido de células madre. Por ejemplo, el oxígeno y los nutrientes difunden a través de la matriz, y cantidades adecuadas deben ser capaces de llegar a las células7. Por otro lado, metabolitos nocivos deben eliminarse lo antes posible, y espacio suficiente para el crecimiento de la célula debe estar disponible7. En consecuencia, las propiedades del material y por lo tanto el tamaño del poro, influyen severamente las posibles beneficio y posibles aplicaciones de la matriz. Dependiendo de las propiedades de los materiales, procesos de crecimiento de la célula diferentes pueden ocurrir en cultura celular 3D, incluyendo la formación de estructuras neuronales; el crecimiento y la diferenciación de las células de la piel o el hueso; y el crecimiento dirigido de líneas celulares especiales, como los hepatocitos o fibroblastos2,3,8,9,10,11. Otro de los puntos crucial que influyen en la posible aplicación de un material es su estabilidad hacia estímulos externos12. Por ejemplo, el hidrogel debe mantener su integridad mecánica en medios de cultivo celular o en el cuerpo humano.
En los últimos años de investigación en 3D de la célula cultura hidrogeles se intensificó, y se llevaron a cabo muchos estudios para resolver la arquitectura 3D de los sistemas13. Hidrogeles compuestos de componentes químicamente sintetizados comúnmente son investigados porque pueden ser sintetizados fácilmente y modificar químicamente y exhiben alta estabilidad (véase Zhu et al., 2011 una revisión)5. Sin embargo, las proteínas tienen muchas propiedades beneficiosas: como supuesto "polímeros de precisión", son biocompatibles; tienen una longitud definida; son relativamente fáciles de modificar; y tienen un gran número de sitios de destino14,15. En este sentido, se pueden generar estructuras altamente específicas, innovadoras para su aplicación en muchos campos. En este estudio, un hidrogel basado en proteína16 fue utilizado para demostrar la capacidad de métodos bien establecidos para influir en la arquitectura 3D del material. Además, también se investigó la capacidad de y la aplicabilidad a la generación de poro.
Existen muchas técnicas diferentes modificar estructuras 3D, incluyendo métodos sencillos y técnicas sofisticadas y altamente especializadas de diversos campos de la ciencia de los materiales. Una técnica generalizada es el uso de electrospinning para generar estructuras bien definidas17. Cargada fibras se tiran de una solución por un campo eléctrico y luego solidifican con la exposición al oxígeno. De esta manera, se pueden producir las fibras en la gama de varios nanómetros hasta varias micras. Técnicas adicionales para ajustar el tamaño, estructura y distribución de los poros dentro de la matriz son la litografía blanda, Fotolitografía, enfoque hidrodinámico, electro-rociado y bio-impresión18,19,20. Un inconveniente importante de estas técnicas es su dependencia de equipos específicos, caros y productos químicos especiales o materiales. Además, experiencia con estas técnicas a menudo no es directamente transferible a los materiales basados en proteína, y muchos de los productos químicos y métodos no son compatibles la célula.
Por otra parte, muchas de las técnicas no se apoyan en equipos especiales, haciéndolos más fácil y más barato aplicar y reproducir. Un método generalizado para la manipulación de la estructura es de fundición solvente21,22,23. Las partículas se agregan antes de la reacción de polimerización y se distribuyen de forma homogénea para saturar la solución. Después de la polimerización, un cambio de condiciones, como una disolución o un cambio de pH, conduce a la solvatación de las partículas, mientras que los poros permanecen dentro del material. Los productos químicos utilizados en estas técnicas, tales como sal, azúcar, parafina, gelatina y tiza, son baratos y fácilmente disponibles. En la liofilización, son congelados hidrogeles hinchados. La sublimación posterior de las fases líquidas bajo un vacío es entonces realizado23,24,25. La sublimación del agua de la red es lo suficientemente suave para mantener las estructuras 3D específicas del material. En el gas que hace espuma, una solución es transmitida con un gas mientras que la polimerización ocurre, dejando poros en el gel de21. El tamaño y la distribución de los poros pueden ajustarse dependiendo de la corriente del gas.
Para formar el proteína del hidrogel, BSA está reaccionada cloruradas tetrakis (hidroximetil) fosfonio (THPC) en una reacción tipo Mannich para permitir la formación de enlaces covalentes entre aminas primarias y los grupos hidroxilo de la molécula de vinculador de cuatro brazos26. Posibles productos intermedios nocivos se eliminan por lavado excesivo del material después de que la reacción ocurra.
Este estudio demuestra la posibilidad de tratar un material a base de BSA con diversas técnicas para manipular y adaptar el tamaño de los poros. Cada una de las técnicas puede utilizarse en cualquier laboratorio a nivel mundial, ya que ningún equipo especial es necesario. Además, diferentes parámetros, tales como relación de hinchazón, degradabilidad enzimática, estabilidad de pH y sensibilidad de la temperatura, fueron examinados y comparados con otras, especialmente respetan a la influencia de las diferentes técnicas en la generación de arquitecturas de 3D. Por último, los materiales fueron funcionalizados con péptidos de célula-adhesivo para investigar la posible aplicación de los materiales para cultivo celular. Se utilizaron dos líneas celulares de diferentes modelos: A549 y MCF7.

<table><tbody><tr><td>Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (high glucose)</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>11140-050</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>10270-106</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>MEM Nonessential Amino Acid Solution</td><td>Sigma Aldrich</td><td>M7145-100ML</td><td></td></tr><tr><td>Trypsin EDTA 0.05% Phenol Red</td><td>Thermo Fisher Scientific</td><td>25300062</td><td></td></tr><tr><td>Ethanol 99.8%, verg&amp;auml;llt</td><td>&amp;Ouml;lfabrik Schmidt</td><td>2133</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Carl Roth&amp;nbsp;</td><td>9265.1</td><td></td></tr><tr><td>Albumin Fraction V</td><td>Carl Roth&amp;nbsp;</td><td>3854.2</td><td></td></tr><tr><td>THPC</td><td>Sigma Aldrich</td><td>404861-100ML</td><td>Toxic</td></tr><tr><td>0.1% Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td>Slightly toxic</td></tr><tr><td>Phalloidin-rhodamine&amp;nbsp;</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>R415</td><td></td></tr><tr><td>3.7% Formaldehyde&amp;nbsp;</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>F8775-25ML</td><td>Toxic</td></tr><tr><td>Rhodamine B</td><td>Sigma Aldrich</td><td>81-88-9</td><td></td></tr><tr><td>Filtropur S 0.2&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>2 83.1826.001&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&amp;micro; slide 8 well</td><td>Ibidi GmbH</td><td>80826</td><td></td></tr><tr><td>KCSSGKSRGDS peptide</td><td>UPEP Ulm</td><td>Custom sysnthesis</td><td></td></tr><tr><td>Ethanol 99.8%, verg&amp;auml;llt</td><td>Carl Roth&amp;nbsp;</td><td>K928.5</td><td></td></tr><tr><td>Falcon 5 ml Polysterene Round-Bottom Tube&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>62.547.254&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Tubes 50 ml&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>62.547.254&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Tubes 1.5 ml&amp;nbsp;&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>72,690,001</td><td></td></tr><tr><td>Tubes 2 ml&amp;nbsp;&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>72,691</td><td></td></tr><tr><td>CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td><td>Greiner</td><td>655073</td><td></td></tr><tr><td>FreezeDryer Epsilon 1-6D,&amp;nbsp;</td><td>Christ, Osterode am Harz, Germany</td><td></td><td></td></tr><tr><td>Confocal Laser Scanning Microscope&amp;nbsp;</td><td>Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484</td><td>Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7</td><td>GSA GmbH</td><td></td><td></td></tr></tbody></table>

easy manipulation of architectures in protein-based hydrogels for cell culture applications

Los hidrogeles son reconocidos como prometedores materiales para aplicaciones en cultura celular debido a su capacidad para proporcionar entornos celulares altamente hidratada. El campo de plantillas 3D está aumentando debido a la semejanza potencial de esos materiales a la matriz extracelular natural. Hidrogeles basados en proteína son particularmente prometedoras porque fácilmente puede ser funcionalizados y puede alcanzar estructuras definidas con propiedades fisicoquímicas ajustables. Sin embargo, la producción de plantillas 3D macroporoso para los usos de la cultura de célula utilizando materiales naturales a menudo está limitada por sus propiedades mecánicas más débiles comparados con los de materiales sintéticos. Aquí, se evaluaron diferentes métodos para producir macroporoso albúmina de suero bovino (BSA)-basado en sistemas de hidrogel, con tamaños de poro ajustable en el rango de 10 a 70 μm de radio. Además, se estableció un método para generar canales de este material basado en proteína que son varias cien micras de largo. Se analizaron los diferentes métodos para producir los poros, así como la influencia del tamaño de poro en material propiedades tales como relación, pH, estabilidad de la temperatura y el comportamiento de la degradación enzimática, la hinchazón. Tamaños de poro se investigaron en el estado nativo, hinchado de los hidrogeles mediante microscopía de láser confocal. La factibilidad de usos de la cultura de célula fue evaluada usando una pegamento celular RGD péptido modificación del sistema de proteína y dos líneas de celular modelo: células de cáncer de mama humano (A549) y adenocarcinomic alveolares basals epitelial las células humanas (MCF7).

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron micr...

Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Diferentes métodos para manipular arquitectura tridimensional en Hidrogeles basados en proteína se evalúan aquí con respecto a las propiedades del material. Las redes macroporoso son funcionalizadas con un péptido de la célula-adhesivo, y se evalúa su viabilidad en cultivo de células utilizando dos líneas celulares de diferentes modelos.

Fácil manipulación de arquitecturas en Hidrogeles basados en proteínas para aplicaciones de la cultura de célula

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

easy-manipulation-architectures-protein-based-hydrogels-for-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

6.7K vistas.  Ulm University. Diferentes métodos para manipular arquitectura tridimensional en Hidrogeles basados en proteína se evalúan aquí con respecto a las propiedades del material. Las redes macroporoso son funcionalizadas con un péptido de la célula-adhesivo, y se evalúa su viabilidad en cultivo de células utilizando dos líneas celulares de diferentes modelos.

Video: Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Plataformas microfabricated para el cultivo celular mecánico dinámico

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Investigación

Bioingeniería

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 ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

Preparación de hidroxi-Paam hidrogeles para desacoplar los efectos de mecanotransducción Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Construcción de Hojas modular de hidrogel para la arquitectura celular 3D a escala macro micropatterned

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Patrones bioactivas proteínas o péptidos en hidrogel con fotoquímica para usos biológicos

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Producción de la proteína elastina como hidrogeles para encapsulación y el Immunostaining de células en 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

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) ...

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 ...