Os autores gostaria de agradecer seu apoio financeiro no quadro "Síntese de Material Bioinspirada" (BioMatS-14) Stiftung Baden-Württemberg.

1. hidrogel preparação
<ol>
	<li>Misture 200 mg de BSA com 1 mL de deionizada H2O para criar ações de BSA de 20% (p/v) (solução estoque A).</li>
	<li>Mix de 165 µ l da solução THPC (134 mg/mL) com 4,835 mL de água deionizada para criar THPC solução (solução B).</li>
	<li>Pesar 1 mg de peptídeo (1,111.1 g/mol) de KCSSGKSRGDS (ou um equivalente do celular-adesivo peptide) e diluí-la em 100 µ l de estéril H2O para obter uma solução de 10 mg/mL (solução C). Nota: Este passo é opcional e só precisa ser incluído se o hidrogel destina-se a aplicação de cultura de células.</li>
	<li>Remova a parte inferior de uma placa de 96 poços e substituí-lo com o envoltório plástico removível. Nota: Para as placas usadas aqui, o fundo pode ser facilmente removido, aplicando pressão no fundo de cada poço; o fundo de plástico fino vai simplesmente cair.</li>
	<li>Solução (B) o estoque Mix 100 µ l de solução-mãe de BSA (A) e 100 µ l de THPC (opcional: 4 µ l de solução-mãe C para funcionalizados, célula-adesivo de hidrogel) da placa de 96 poços para obter 200 µ l de hidrogel. Misture os componentes pipetando acima e para baixo pelo menos 5 vezes a garantia de um hidrogel uniforme após a polimerização.</li>
	<li>Coloque a placa de 96 poços à temperatura ambiente (RT) por cerca de 10 min até hidrogel todos corretamente é polimerizadas.</li>
	<li>Lentamente e com cuidado Retire o plástico da parte inferior da placa.</li>
	<li>Pressione o hidrogel fora a placa de 96 poços, usando um pequeno selo e transferi-los para tubos de 1,5 mL com PBS estéril, pH 7,4. Nota: O hidrogel tem uma forma cilíndrica, com um diâmetro de aproximadamente 4 mm e uma altura de 8 mm.</li>
	<li>Armazene o hidrogel em tampão fosfato salino (PBS) a 4 ° C por até diversos meses.</li>
</ol>
2. o hidrogel de liofilização
<ol>
	<li>Encher tubos de 1,5 mL com 500 µ l de deionizada estéril H2O e retire a tampa. Transferi o hidrogel aos tubos de reação de 1,5 mL, usando uma espátula.</li>
	<li>Envolva os tubos de 1,5 mL firmemente com parafina filme-pelo menos três camadas para cada frasco. Use uma agulha para perfurar furos pequenos no filme para permitir a liberação de gás do tubo.</li>
	<li>Proceda de uma das seguintes etapas:
	<ol>
		<li>Transferi o frasco para solução de nitrogênio líquido por 5 min garantir que a água e hidrogel congelam completamente. Imediatamente após a remoção do nitrogênio líquido, transferi os frascos para o secador para evitar que o material de descongelamento de gelo. Nota: Este procedimento resulta em poros sobre 10-15 µm no raio.</li>
		<li>Manter os frascos a-20 ° C durante a noite para congelar lentamente o hidrogel. Imediatamente após a remoção de-20 ° C, transferi os frascos para o secador para evitar que o material de descongelamento de gelo. Nota: Este procedimento resulta em poros sobre 50-60 µm no raio.</li>
	</ol>
	</li>
	<li>Após 24h (e a evaporação completa da água e em torno do hidrogel), descongelar o material por retirando-o secador de gelo. Nota: O tamanho dos poros pode ser analisado com laser confocal, microscopia (etapa 5, "Visualização de hidrogel").</li>
</ol>
3. partícula lixiviação
<ol>
	<li>Prepare o hidrogel como descrito nos passos 1.1-1.5.</li>
	<li>Diretamente após a mistura dos componentes, adicione NaCl até que ocorra a saturação (36 mg/mL). Adicione sal até um cristal de sal pode ser visto na solução como um precipitado branco.</li>
	<li>Transferir a placa de 96 poços para uma coqueteleira e agite bem até polimerização ocorre (cerca de 10 min). Remova o hidrogel da placa, conforme descrito nas etapas 1.7-1.8.</li>
	<li>Incube o hidrogel pelo menos 24 h a RT em água estéril num agitador (20 rpm) para eluir todo sal a partir do modelo de hidrogel. Armazene o hidrogel a 4 ° C em PBS por até vários meses.</li>
</ol>
4. formação do canal
<ol>
	<li>Prepare o hidrogel conforme descrito na etapa 1. Remover um hidrogel de solução usando uma pinça, remova o excesso de água com papel altamente absorvente e colocá-lo em cima de um bloco de gelo seco.</li>
	<li>Congelar o hidrogel por 30 s e remova-a cuidadosamente do bloco. Não danifique o hidrogel; cuidadosamente com uma espátula para raspar o bloco. Transfira o hidrogel para um tubo de 1,5 mL e secá-lo durante a noite a 37 ° C.</li>
</ol>
5. hidrogel visualização
<ol>
	<li>Prepare uma solução stock de rodamina B diluindo-se 1 mg de rodamina B em 10 mL de PBS. Preparar uma diluição serial diluindo a solução-mãe de rodamina em PBS até atingir uma concentração de 0,001 mg/mL (factor de diluição: 100)</li>
	<li>Remover o hidrogel da solução de armazenamento (por exemplo, da etapa 2.4.) e transferir para um tubo de 1,5 mL com 1 mL de solução de rodamina B 0,01 mg/mL. Mancha o hidrogel durante a noite em solução de rodamina B a RT</li>
	<li>No dia seguinte, transferi o hidrogel que é para ser visualizado a 10 mL de PBS (pH 7,4) e lavagem pelo menos 3 h.</li>
	<li>Transferir o hidrogel numa µ-lâmina 8 bem e cubra com PBS.</li>
	<li>Pequenas fatias para fora o hidrogel (cerca de 0,5 mm em altura), usando uma lâmina de corte.</li>
	<li>Usando um laser confocal microscópio, Visualizar o hidrogel no comprimento de onda de 514 nm (objectivo: CE plano-Neofluar 40 x / 1,30 óleo DIC M27, modo de varredura de avião: 514 nm e zoom: 1 X).</li>
</ol>
6. viabilidade de cultura de célula
<ol>
	<li>Estéril-filtro todos estoque de soluções (A, B e C) com um filtro de 0,45 µm.</li>
	<li>Prepare o hidrogel como descrito nos passos 1.1-1.4, incluindo o peptídeo de célula-adesivo antes da polimerização de hidrogel.</li>
	<li>Após a mistura dos componentes, pipete imediatamente a mistura para um µ-slide 8 bem até que o fundo é completamente coberto. Nota: Esta etapa deve ser executada rapidamente e diretamente após a mistura dos componentes, como polimerização ocorre dentro de minutos no slide.</li>
	<li>Células de adesão de cultura e passagem para obter uma suspensão de célula única, utilizando técnicas de cultura de célula padrão. Transferi as células previamente aquecido, estéril DMEM celular cultura suplementado com soro fetal bovino (FBS, 10% (p/v)), penicilina-estreptomicina (1% (p/v)) e solução de aminoácido não essencial (MEM, 1% (p/v)).</li>
	<li>Conte as células com um Neubauer contando câmara e cuidadosamente Pipetar 200 µ l do número desejado de células (2 x 105 células/cm2) sobre a superfície de hidrogel.</li>
	<li>Cobrir o µ-slide 8 bem com a tampa e transferi-lo para uma incubadora (37 ° C, 5% CO2). Incubar durante pelo menos 4 h a 37 ° C.</li>
	<li>Após pelo menos 4 h de ligação celular, lave as células duas vezes com 200 µ l de cultura de células estéreis PBS.</li>
	<li>Consertar as células com 200 µ l de formaldeído de 3,7% (v/v) durante 10 minutos a RT e lave duas vezes com PBS (~ 200 µ l). Nota: Usar equipamento de protecção adequado ao manusear o formaldeído.</li>
	<li>Permeabilize as células com 200 µ l de 0.1% Triton X 5 min. Lave duas vezes com PBS.</li>
	<li>Manche as células com rodamina-faloidina misturando 5 µ l de estoque metanólica com 195 µ l de PBS e adicioná-lo às células para RT. mancha as células por 20 min no escuro. Lave duas vezes com PBS.</li>
	<li>Investigar as propriedades de aderência de célula usando um microscópio confocal em 514 nm (objectivo: CE plano-Neofluar 40 x / 1,30 óleo DIC M27, modo de varredura de avião: 514 nm e zoom: 1 X). Analisar as imagens usando o software apropriado (consulte a Tabela de materiais).</li>
</ol>
7. hidrogel Propriedades
<ol>
	<li>Relação de inchaço.
	<ol>
		<li>Seque completamente o hidrogel a 37 ° C pelo menos um dia.</li>
		<li>Pesar cada hidrogel e anotar o peso exato.</li>
		<li>Encha um tubo de reação de 2 mL com 1,5 mL de PBS.</li>
		<li>Transferir o hidrogel neste tubo de reação de 2ml e mergulhe-a completamente em PBS.</li>
		<li>Deixe o hidrogel para pelo menos dois dias em PBS em RT para atingir o equilíbrio com a PBS.</li>
		<li>Depois de três dias, retire o hidrogel da solução e seque-o com um lenço de papel para remover o excesso de água da superfície de hidrogel.</li>
		<li>Pese o hidrogel.</li>
		<li>Calcule a relação inchaço usando a seguinte fórmula: 
		<img alt="Equation" src="/files/ftp_upload/55813/55813eq1.jpg"> onde Wd é o peso do gel seco (etapa 7.1.2.) e Ws é o peso do gel molhado (etapa 7.1.7). Multiplica com 100 para obter a porcentagem de relação a inchaço.</li>
	</ol>
	</li>
	<li>estabilidade de pH e temperatura.
	<ol>
		<li>Transfira 5 mL de PBS para um tubo de reação de 15 mL.</li>
		<li>Ajuste a solução ao pH adequado (por exemplo, pH 2, 7 ou 10) com NaOH e HCl. Bring a solução à temperatura adequada (por exemplo, RT 37 ° C e 80 ° C).</li>
		<li>Transferi o hidrogel que está a ser investigado por sua estabilidade para a solução adequada. Se necessário, reajuste o pH. Nota: Todos hidrogel deve ser completamente inchados neste ponto (veja a relação de inchaço) para evitar a interpretação incorreta do peso devido ao inchaço do material.</li>
		<li>Após determinados intervalos de tempo, retire o hidrogel da solução (por exemplo, cada hora durante 2 dias), seque-o com um lenço de papel altamente absorvente para remover o excesso de água e pesá-lo.</li>
	</ol>
	</li>
	<li>Degradação enzimática.
	<ol>
		<li>Prepare uma solução de enzima de estoque de 300 tripsina U e pepsina, conforme as instruções do fabricante.</li>
		<li>Transferi o hidrogel (por exemplo, da etapa 1.8) para a solução adequada. Nota: Todos os hidrogel deve ser completamente inchados neste momento para evitar a interpretação incorreta do peso.</li>
		<li>Após determinados intervalos de tempo, retire o hidrogel da solução (por exemplo, cada hora), seque-o com um lenço de papel altamente absorvente para remover o excesso de água e pesá-lo.</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>

Os autores declaram que eles têm não tem interesses financeiro concorrente.

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...

Hidrogel é materiais que formam redes 3D insolúveis capaz de ligação a grandes quantidades de água. Tais materiais podem fornecer excelentes condições ambientais para as células vivas. Atualmente, existe uma crescente interesse na geração de estruturas tridimensionais de hidrogel e no desenvolvimento de processos para adequar suas propriedades químicas e físicas. Uma vez que este é alcançado, um modelo para o crescimento das células e a manipulação do comportamento celular pode ser gerado1,2,3,4. Estas estruturas 3D não só criam um ambiente mais natural e realista do que abordagens convencionais bidimensionais, mas eles também revelar novas possibilidades para o crescimento de células-tronco ou tumor modelos5. Diferentes materiais possuem uma gama de características que principalmente depende do tamanho dos poros do gel de6. Os poros desempenham um papel crucial em aplicações de cultura de células, a engenharia de tecidos e o crescimento direcionado de células-tronco. Por exemplo, oxigênio e nutrientes difundem pela matrix, e quantidades adequadas devem ser capazes de atingir as células7. Por outro lado, metabólitos prejudiciais devem ser removidos logo que possível, e espaço suficiente para o crescimento da célula deve ser disponível7. Consequentemente, as propriedades do material e, portanto, o tamanho dos poros, influenciam severamente os potencial benefício e possível aplicações da matriz. Dependendo as propriedades do material, processos de crescimento de células diferentes podem ocorrer em cultura de células 3D, incluindo a formação de estruturas neuronais; o crescimento e diferenciação das células da pele ou osso; e o crescimento direcionado de linhas de células-tronco especiais, como hepatócitos ou fibroblastos2,3,8,9,10,11. Outro ponto crucial, influenciando a possível aplicação de um material é a sua estabilidade no sentido de estímulos externos12. Por exemplo, o hidrogel deve manter sua integridade mecânica em meios de cultura celular ou o corpo humano.
Nos últimos anos, pesquisas sobre cultura de células 3D hidrogel intensificou-se, e muitos estudos foram realizados para resolver as arquiteturas 3D dos sistemas13. Hidrogel composto por componentes quimicamente sintetizados mais comumente é investigados, porque podem ser facilmente sintetizados e quimicamente modificados e eles apresentam alta estabilidade (veja Zhu et al, 2011 para uma revisão)5. No entanto, as proteínas têm muitas propriedades benéficas: tão chamados "polímeros de precisão", eles são biocompatíveis; Eles têm um comprimento definido; Eles são relativamente fáceis de modificar; e eles têm um grande número de sites de destino14,15. A este respeito, estruturas inovadoras, altamente específicas podem ser geradas para aplicação em muitos campos. Neste estudo, um hidrogel à base de proteína de16 foi usado para demonstrar a capacidade de bem estabelecidos métodos para influenciar a arquitetura 3D do material. Além disso, a capacidade de e aplicabilidade para geração de poros também foi investigada.
Muitas técnicas diferentes estão disponíveis para modificar estruturas 3D, incluindo ambos os métodos simples e técnicas sofisticadas, altamente especializadas de diferentes áreas da ciência de materiais. Uma técnica comum é o uso de eletrofiação para gerar estruturas bem definidas17. Carregada de fibras são puxadas a partir de uma solução por um campo elétrico e em seguida solidificam após a exposição ao oxigênio. Desta forma, podem ser produzidas fibras no intervalo de vários nanômetros até alguns mícrons. Técnicas adicionais para ajustar o tamanho, estrutura e distribuição dos poros dentro da matriz são macia litografia, fotolitografia, focalização hidrodinâmica, electro-pulverização e bio-imprimir18,19,20. Uma desvantagem significativa dessas técnicas é sua dependência específico, caros equipamentos e produtos químicos especiais ou materiais. Além disso, a experiência com estas técnicas, muitas vezes não é diretamente transferível para materiais à base de proteínas, e muitos dos produtos químicos e métodos não são celulares compatíveis.
Por outro lado, muitas técnicas não se baseará equipamento especial, tornando-os mais fácil e mais barato para aplicar e reproduzir. Um método generalizado para manipulação de estrutura é solvente fundição21,22,23. As partículas são adicionadas antes da reação de polimerização e são distribuídas homogênea até para saturar a solução. Após a polimerização, uma mudança de condições, tais como uma diluição ou uma alteração de pH, leva para a solvatação das partículas, enquanto os poros permanecem dentro do material. Os produtos químicos utilizados nestas técnicas, tais como sal, açúcar, parafina, gelatina e giz, são baratas e prontamente disponíveis. Na liofilização, hidrogel inchadas está congelados. A sublimação subsequente das fases líquidas sob um vácuo é então realizada23,24,25. Sublimação da água da rede é suficientemente suave para manter as estruturas 3D específicas do material. Em gás de espuma, uma solução é transmitida com um gás durante a polimerização ocorre, deixando os poros dentro do gel de21. O tamanho e a distribuição dos poros podem ser ajustadas dependendo do fluxo de gás.
Para formar a proteína hidrogel, BSA é reagido com cloreto de tetrakis (hidroximetil) do phosphonium (THPC) em uma reação de Mannich-tipo para permitir a formação de ligações covalentes entre aminas primárias e os grupos hidroxi de 4 braços vinculador molécula26. Possíveis intermediários prejudiciais são removidos por lavagem excessiva do material, depois que a reação ocorre.
Este estudo demonstra a possibilidade de tratar de um material à base de BSA com diferentes técnicas para manipular e ajustar o tamanho dos poros. Cada uma das técnicas pode ser usada em qualquer laboratório em todo o mundo, como nenhum equipamento especial é necessário. Além disso, diferentes parâmetros, tais como inchaço ratio, degradabilidade enzimática, estabilidade do pH e sensibilidade à temperatura, foram examinados e comparados uns aos outros, especialmente respeitam à influência das diferentes técnicas na geração de arquiteturas 3D. Finalmente, os materiais foram acrescidos com peptídeos de célula-adesivo para investigar a possível aplicação dos materiais para cultura de células. Utilizaram-se duas linhas de célula diferente modelo: A549 e 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

Hidrogel é reconhecidos como materiais promissores para aplicações de cultura de células devido à sua capacidade para fornecer ambientes de célula altamente hidratado. O campo de modelos 3D está a aumentar devido à semelhança potencial desses materiais para a matriz extracelular natural. Hidrogel à base de proteínas é particularmente promissor, porque pode facilmente ser acrescidas e pode atingir estruturas definidas com propriedades físico-químicas ajustáveis. No entanto, a produção de modelos 3D de se para aplicações de cultura de células utilizando materiais naturais é muitas vezes limitada pela suas propriedades mecânicas mais fracas em comparação com as de materiais sintéticos. Aqui, diferentes métodos foram avaliados para produzir se albumina de soro bovino (BSA)-com base em sistemas de hidrogel, com tamanhos de poros ajustável na faixa de 10 a 70 µm no raio. Além disso, estabeleceu-se um método para gerar canais neste material à base de proteínas que são vários cem mícrons longos. Os diferentes métodos para produzir os poros, bem como a influência do tamanho dos poros em propriedades materiais, tais como inchaço ratio, pH, estabilidade de temperatura e comportamento de degradação enzimática, foram analisados. Tamanho dos poros foram investigado no estado nativo, inchado do hidrogel usando laser confocal, microscopia eletrônica de varredura. A viabilidade para aplicações de cultura de células foi avaliada com célula-adesivo RGD peptídeo modificação do sistema de proteína e duas linhas de célula modelo: células de câncer de mama humano (A549) e adenocarcinomic alveolares basais epiteliais 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 a arquitetura tridimensional em hidrogel à base de proteínas são avaliados aqui com respeito a propriedades do material. As redes se são acrescidas com um peptídeo de célula-adesivo, e sua viabilidade na cultura de pilha é avaliada usando duas linhas de células de modelo diferente.

Fácil manipulação das arquiteturas em hidrogel à base de proteínas para aplicações de cultura de células

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

Research

JoVE Journal

Biochemistry

6.7K visualizações.  Ulm University. Diferentes métodos para manipular a arquitetura tridimensional em hidrogel à base de proteínas são avaliados aqui com respeito a propriedades do material. As redes se são acrescidas com um peptídeo de célula-adesivo, e sua viabilidade na cultura de pilha é avaliada usando duas linhas de células de modelo diferente.

Vídeo: 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 cultura de células mecanicamente dinâmico

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

Bioengenharia

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.

Fabricação de hidrogéis micropatterned para Sistemas de Cultura Neural usando fotolitografia Projeção Dinâmica 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.

Preparação de Hidroxi-PAAm hidrogéis para dissociar os efeitos de Mecanotransdução Sugestões

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.

Construção de Fichas de hidrogel modular para micropadronadas Macro-escalado 3D Cellular Arquitetura

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.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura 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.

Sintonizável hidrogéis a partir de Pulmonar Matriz Extracelular para Cultura Celular 3D

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.

Padronização bioativas proteínas ou peptídeos em hidrogel usando fotoquímica para aplicações biológicas

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.

Produção de elastina, como proteína hidrogel para encapsulamento e imunocoloração de células em 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.

Medição de metaloproteinase matriz celular Global e atividade metabólica em hidrogel 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.

Tensão controlada de hidrogéis 3D sob imagem de microscopia ao vivo

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