This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1. Ativando a superfície de lamelas de vidro <ol><li> Lugar de vidro lamelas circulares (25 mm de diâmetro) em uma placa de Petri e smear solução de NaOH 0,1 M nele por 5 min (Cobertura de vapores químicos recomendado). </li><li> Remover a solução de NaOH e mergulhar totalmente lamelas com DDH 2 O estéril por 20 min enquanto balançando suavemente sobre uma placa de balanço em uma capa de cultura estéril. </li><li> Escorra estéril DDH 2 O e repita o passo 1.2. </li><li> Retirar as lamelas com pinças estéreis e colocá-los em uma nova placa de Petri com a face para cima ativado. </li><li> Lamelas secas sob um fluxo constante de elevada pureza do gás de azoto. </li><li> Num capuz de cultura estéril, esfregaço de uma fina camada de acrilato de 3 (trimetoxissilil) propilo (92%) do lado da lamela activado durante 1 h. </li><li> Extensivamente lavar lamelas de vidro com 3 lavagens de DDH 2 O estéril e mergulha-as na DDH 2 O estéril em uma nova placa de Petri. </li><li> Toque na placa de Petricom Parafilm e colocá-lo em uma placa oscilante sob agitação suave durante 10 min. </li><li> Retirar as lamelas DDH 2 O, com uma pinça estéreis com pontas finas e coloque-os em uma nova placa de Petri com a face para cima ativado. </li><li> Armazenar à temperatura ambiente, em local seco com papel alumínio para evitar que a poeira grude as lamelas. </li></ol> 2 Preparação de Hidroxi-PAAm hidrogéis <ol><li> Prepara-se uma massa de 65 mg de acrilamida de N-hidroxietil (HEA) num tubo Eppendorf de 1,5 ml. É importante para preparar uma solução de HEA fresco. </li><li> Adicionar 1 ml de tampão HEPES 50 mM de HEA e misture utilizando um agitador até à dissolução completa do HEA. </li><li> Adicionar 400 ul de 40% w / w em solução de HEPES acrilamida e o volume necessário de 2% w / w em solução de HEPES bis-acrilamida (ver Tabela 1) para atingir a rigidez do hidrogel desejada. Ajustar com HEPES 50 mM a um volume final de 5 ml. </li><li> Misture a solução com um agitador e degas TInuma câmara de vácuo durante 20 minutos, a fim de reduzir a concentração de oxigénio na solução, que evita a polimerização hidroxi-PAAm. </li><li> Dentro de uma câmara estéril, filtrar a solução desgaseificados com um filtro de 0,2 um de tamanho de poro, de modo a esterilizar. </li><li> Ative lamínulas de vidro circulares (22 mm de diâmetro), em um aspirador de UV / ozônio durante 7 min. </li><li> Prepare a 100 ul de solução de persulfato de amónio (APS) 10%, que é de 10 mg de EPA 100 ul ddH 2 O. É importante para preparar uma solução de APS fresco. </li><li> Adicionar 2,5 mL de tetrametilenodiamina (TEMED) e 25 ul de solução de APS ao hidroxi-PAAm solução esterilizada (passo 2.5), para iniciar a polimerização. Misture a solução por 3 pipettings sucessivas sem introdução de bolhas, em condições estéreis. </li><li> Dentro de uma câmara esterilizada, colocar uma gota de 25 ul da solução de hidroxi-PAAm sobre uma lamela de 25 mm (disponível a partir do passo 1.9) e colocar imediatamente a gl 22 milímetroslamela de burro (preparado no passo 2.5) em cima da gota para espremer a solução de hidroxi-PAAm. Centralize a lamela de vidro 22 mm com pinças estéreis e eliminar possíveis bolhas. </li><li> Permitir hidrogéis PAAm-hidroxi para polimerizar à temperatura ambiente durante 15 min. Inverta manualmente a solução restante hidroxi-PAAm no tubo Eppendorf a seguir à realização do processo de polimerização. </li><li> Mergulhar totalmente lamelas com ddH2O estéril, e cuidadosamente separar as lamelas 22 mm de vidro através da introdução da extremidade de uma lâmina de barbear entre as lamelas de 22 mm de vidro e a camada de hidrogel hidroxi-PAAm. </li><li> Lave hidrogéis-PAAm hidroxi com PBS estéril (3 trocas de PBS) e deixe o gel totalmente imersos em PBS estéril para manter a hidratação. </li><li> Armazenar hidrogéis PAAm-hidroxi em PBS estéril, a 4 ° C durante até 3 dias. </li></ol> 3. polidimetilsiloxano (PDMS) Microstamp Fabrication NOTA: A fabricação de um mestre de silício é necessária antes de start o PDMS fabricação microstamp. Este microfabricação de um mestre de silício pode ser feito por meio de técnicas de litografia, que requer equipamentos e treinamento especializado. Colaborações com uma instalação de nanofabricação são incentivados a fabricar o mestre de silício. Alternativamente, entre em contato com a empresa que fabrica os mestres de silício microestruturada feitos sob demanda. É importante notar que a fabricação do mestre de silício só precisa ser feito uma vez. Na verdade, os mestres de silício microstructured pode ser usado indefinidamente para produzir selos elastoméricos. <ol><li> Mistura de PDMS e o agente de cura em um proporção de 10: 1, num copo de plástico e homogeneizar utilizando uma pipeta de 10 min. </li><li> Desgaseificar a mistura PDMS sob vácuo para remover as bolhas de ar que se formaram durante o passo 3.1. </li><li> Coloque o mestre de silício microestruturada em uma placa de Petri e lançou a 10 mm de espessura da mistura de PDMS desgasado nele sem formar bolhas. </li><li> Let curar o PDMS durante 2 horas a 60 ° C numaforno. </li><li> Em um ambiente livre de poeira, peel-off da camada de PDMS e impostos especiais de consumo 1 cm 2 microstamps com um bisturi. </li><li> Utilizando uma pinça, coloque PDMS microstamps padrão-up em uma placa de Petri. </li></ol> 4. micropatterning Hidroxi-PAAm hidrogéis <ol><li> Colocar solução PDMS microstamps em uma mistura de etanol / água (50/50) e sonicado durante 15 minutos. </li><li> Secar os selos com uma corrente de fluxo de azoto, e colocá-los padrão-se num dispositivo de limpeza de UV / Ozono (λ &lt;200 nm) durante 7 min. </li><li> Dentro de uma câmara esterilizada, colocar uma gota de 150 mL de uma solução de proteína desejada (por exemplo, 100 ug / ml de laminina em PBS ou 25 ug / ml de fibronectina em PBS) para a superfície de uma microestrutura de 1 cm 2 de PDMS selo. </li><li> Opcional: modificar a concentração da solução de proteínas para modular a densidade celular-ligando. </li><li> Espalhar a solução de proteína através da superfície do selo, movendo-a com a ponta de uma pipeta estéril para cada cantoo selo. </li><li> Deixar a solução de proteína para adsorver sobre o selo PDMS durante 60 minutos sob uma cobertura estéril. Desligue lâmpadas para evitar danos proteína. </li><li> Dentro de uma câmara estéril, transferem-hidroxi PAAm lamelas revestidas (disponíveis a partir do passo 2.13) em uma placa de Petri. </li><li> Remover o excesso de PBS a partir da superfície de substratos hidroxi-PAAm com um fluxo baixo de azoto sob condições estéreis. Parar o processo logo que há evidência de água que está sobre a superfície do gel é observado. O gel não deve ser completamente seca nesta fase. </li><li> Secar cuidadosamente a superfície estruturada do PDMS selo com um fluxo constante de elevada pureza do gás de azoto. </li><li> Segure o selo revestido de proteína com uma pinça de vestir e colocar a superfície estruturada em contato com a superfície do hidrogel seco. Aplicar os pontos de pressão breves com a ponta da pinça sobre a parte superior do selo PDMS para assegurar um bom contacto entre as microestruturas do selo e a superfície do hidrogel. </li><li> Deixe o selo PDMS em the superfície do hidrogel durante 1 hora à temperatura ambiente. </li><li> Remova cuidadosamente PDMS selos a partir de hidrogéis hidroxi-PAAm com uma pinça de vestir e seguir o passo 4.1 para limpar o selo. </li><li> Lave exaustivamente os hidrogéis-PAAm hidroxi estampadas por 3 trocas de PBS (pH = 7,4) em condições estéreis por 10 minutos por troca. </li><li> Opcional: micropadrões adicionais de outras proteínas de ECM podem ser adicionados ao PAAm hidroxi-superfície, seguindo passos 4,5-4,11. </li><li> As zonas não impressas passivar com uma solução estéril de BSA a 5 mg / ml em PBS durante uma noite a 4 ° C, sob uma agitação suave em uma placa de balanço. </li><li> Lave extensivamente por 3 trocas de PBS (pH = 7,4) em condições estéreis por 10 minutos por troca. Nesta fase, os hidrogéis-hidroxi PAAm estampadas podem ser armazenadas a 4 ° C até uma semana. </li></ol> 5. Deposição celular em micropatterned hidroxi-PAAm hidrogéis <ol><li> Incubar lamelas em mídia celular para 30-45 min antes de chapeamento cvaras. </li><li> Lave as células aderentes cultivadas num frasco de 2 75 centímetros cultura com PBS estéril a 37 ° C e separá-lo com 3 ml de tripsina-EDTA ou Accutase durante 10 min. </li><li> Transferir a quantidade desejada de pré-aquecido completa de meio de crescimento apropriado para a sua linha de células para dentro do frasco contendo as células destacadas e centrifugar a suspensão de células durante 3 minutos a 650 x g. </li><li> Retirar o sobrenadante com uma micropipeta e ressuspender as células em meio de cultura completo às 15-20,000 células / ml. </li><li> Adicionar 4 ml da solução de células para uma lamela micropadronadas (obtido a partir do Passo 5.1) e colocar a lamela coberta por células em cultura numa incubadora a 37 ° C e 5% de humidade durante 1-2 horas. </li><li> Aspirar delicadamente as células solto e substituir meio de cultura. Devolver as células ligadas à incubadora e deixá-los totalmente espalhar (3-6 horas, dependendo do tipo de célula). </li></ol>

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No conflicts of interest declared.

Muitos em observações in vitro em moderna biologia celular ter sido realizada em lamelas de vidro rígidas, muitas vezes revestidos com uma fina camada de proteínas de ECM ou de péptidos sintéticos contendo a sequência RGD. No entanto, tais substratos de cultura básicos não recapitular toda a complexidade físico-química da ECM e, portanto, não fornecem um modelo exato para o estudo de processos Mecanotransdução celulares. Para resolver este problema, propomos uma alternativa simples para f...

Muitos aspectos do microambiente celular local (por exemplo, rigidez, tamanho de poro, a natureza das proteínas, ou a densidade de célula-ligando) fornecer um conjunto de coordenadas de sinais de regulação que controlam os processos celulares tais como a motilidade, a proliferação celular, diferenciação, e a expressão do gene. As modificações das propriedades físico-químicas do ambiente extracelular pode ser percebida por células e provocar consequências fisiológicas diferentes, incluindo deformação da polarização celular, migração, e diferenciação. Ainda não está claro, no entanto, como as células traduzir modificações ECM em sinais bioquímicos celulares. Por isso, é de grande importância para engenheiro controlado em microambientes in vitro que podem reproduzir as interações entre células e seu microambiente para estudar vias Mecanotransdução. Para resolver este problema, que recentemente introduziu um novo método 1, chamado de hidrogéis-PAAm hidroxi, para gerar facilmente de dois centavomatrizes nsional suaves que permitem controlar de forma independente pistas Mecanotransdução importantes: a rigidez da matriz, geometria celular e confinamento, da natureza da proteína e densidade de células-ligante. MEC orienta processos celulares através de gradientes em morphogens (quimiotaxia), proteínas adesivas (haptotaxia) e rigidez (durotaxis). Ao longo das últimas décadas, avançado em plataformas in vitro têm sido desenvolvidos para isolar esses sinais extracelulares, a fim de trazer à tona como as células são capazes de traduzir características bioquímicas e biofísicas em processos fisiológicos 2-5. Foram desenvolvidos por feixe de elétrons 6, 7 fotolitografia, imobilização fotoquímica de 8, ou técnicas assistidos por plasma 9 para dirigir o crescimento de células vivas nos substratos micropatterned. Embora essas técnicas tiveram resultados importantes, a maioria deles não permitem a discriminação entre a influência individual de diferentes pistas sobre o comportamento das célulase requerem instalações técnicas que alguns laboratórios podem pagar. Entre estas técnicas, a impressão microcontact (μCP), tem surgido como um método robusto e acessível para a criação de células-adesiva micro-ilhas 10. Mais recentemente, os esforços extensos 11-14 foram feitos para desenvolver hidrogéis com μCP em rigidez ajustáveis, a fim de reproduzir a vasta gama de rigidez observados em tecidos vivos. Entre essas obras, poliacrilamida (PAAm) tornou-se popular 15 e já é uma das matrizes à base de polímeros mais utilizados para estudos biomecânicos celulares ensaios. PAAm superfícies são geralmente funcionalizada com o agente de reticulação heterobifuncional proteínas-sulfossuccinimidil-6-N [4&#39;-azido-2&#39;-nitrophenylamido] (sulfo-SANPAH) e de ECM são ligados à superfície através da activação de UV do nitrofenil sulfo-SANPAH grupos azida 16. Uma outra técnica consiste no acoplamento de hidrazina para proteínas que têm sido severamente oxidadocom periodato 17. Hynd e colaboradores introduzida uma técnica para padronizar as superfícies de hidrogel biomiméticos com proteínas e peptídeos que requer a fotopolimerização na presença de um monómero acroyl-estreptavidina 18. Mais recentemente, Tseng et al. Relataram um novo método micropatterning 19 com base na exposição à radiação UV profunda de PAAm através de uma máscara de quartzo óptico que requer a incubar geles PA activado com 1-etil-3-[3-dimetilaminopropil] carbodiimida (EDC) e soluções aquosas de N-hidroxissuccinimida (NHS) antes de adicionar a proteína. Apesar da capacidade destas técnicas para criar proteínas homogéneas e reprodutíveis micropadrões, a maior parte deles sofrem grandes limitações: os processos de síntese de longo (por exemplo, a diálise, liofilização, etc), compostos químicos caros (por exemplo, ácido hialurónico, sulfo-SANPAH) ou UV profundo irradiação. Além disso, estas técnicas não permitem a modulação independente de rigidez do substrato, micropatterngeometria, ECM natureza proteica, e a densidade celular-ligando. Tendo em conta estas limitações, temos desenvolvido uma abordagem base de acrilamida e novos simples que permite a imobilização de uma variedade de proteínas e biomoléculas em hidrogéis macios e permite sintonia independente de sinais Mecanotransdução a fim de decifrar o seu papel em funções celulares. Em vez de tratar hidrogéis PAAm com compostos químicos agressivos, introduzimos um monômero acrilamida comercial com grupos hidroxila durante a polimerização PAAm. Esta operação simples supera a propriedade anti-aderente intrínseca de hidrogéis PAAm sem quaisquer outros requisitos técnicos. A presença de grupos hidroxilo conduz a uma elevada afinidade de hidrogéis PAAm-hidroxi para proteínas e biomoléculas que formam interacções hidrogénio por entrelaçamento. Em combinação com μCP, hidrogéis-hidroxi PAAm permitir a rápida geração de plataforma cultura bidimensional com um controlo independentesobre a rigidez da matriz, o tipo de proteínas de ECM, a densidade celular ligando-confinado e adesividade, que está previsto para ser uma plataforma poderosa para o estudo Mecanotransdução. O objectivo deste protocolo é fornecer as informações necessárias para a tomada facilmente hidrogéis-PAAm hidroxi sem qualquer experiência em ciências de materiais. O objetivo final é fornecer um meio para que os pesquisadores fazem perguntas fisiologicamente relevantes nos níveis celulares e teciduais que podem levar a uma melhor compreensão das vias Mecanotransdução envolvidas em mecanismos fisiopatológicos.

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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
			<td style="width:71px;">Ultra-Violet Products</td>
			<td style="width:119px;">Model PR-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
			<td style="width:119px;">Model KS 130 Basic</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Vortexer</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
			<td style="width:71px;">Applied Vacuum Engineering</td>
			<td style="width:119px;">DP- 8-KIT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dressing tissue forceps</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F4392-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5731-500EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminium foil, thickness 0.5&#160;mm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">266574-3.4G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopore membrane filter (0,2 &#181;m pore size)</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">GTTP Filter code</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (25 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z114820</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Protein microcentrifuge tubes</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel handles</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2896-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel blades</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2771-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cell culture flasks (75 cm2)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ultrasonic bath tray, solid (stainless steel)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z613983-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acrylamide (powder)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3553</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">146072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N-Hydroxyethylacrylamide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">697931</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3678</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimetoxysilyl)propyle acrylate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1805</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L2020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Endothelial cell growth medium</td>
			<td style="width:71px;">Cells Applications</td>
			<td style="width:119px;">211K-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues

Está agora bem estabelecido que muitas funções celulares são reguladas por interacções de células com pistas físico-químicas e mecânicas do seu ambiente de matriz extracelular (ECM). As células eucarióticas sentir constantemente seu microambiente local através mechanosensors de superfície para a transdução de mudanças físicas de ECM em sinais bioquímicos, e integrar esses sinais para alcançar mudanças específicas na expressão gênica. Curiosamente, os parâmetros físico-químicas e mecânicas do casal ECM lata uns com os outros para regular o destino celular. Portanto, a chave para a compreensão Mecanotransdução é dissociar a contribuição relativa dos sinais de ECM sobre as funções celulares. Aqui apresentamos um protocolo experimental detalhado para gerar rapidamente e facilmente hidrogéis biologicamente relevantes para a afinação independente de pistas Mecanotransdução in vitro. Nós, os hidrogéis de poliacrilamida quimicamente modificados (Paam) para ultrapassarem as suas intrinsecamente não-Adhesive propriedades, incorporando monómeros de acrilamida funcionalizado com hidroxilo, durante a polimerização. Obtivemos um romance PAAm hidrogel, chamado hidroxi-PAAm, que permite a imobilização de qualquer natureza desejada das proteínas da MEC. A combinação de hidroxi-PAAm hidrogéis com impressão microcontact permite controlar de forma independente a morfologia das células individuais, a rigidez da matriz, a natureza e a densidade das proteínas da MEC. Nós fornecemos um método simples e rápido que pode ser configurado em cada laboratório de biologia para estudar em processos Mecanotransdução células in vitro. Nós validar este romance plataforma bidimensional através da realização de experimentos em células endoteliais que demonstram uma ligação mecânica entre rigidez ECM eo núcleo.

A Figura 1A apresenta a co-polimerização de acrilamida (AAm) e bisacrilamida (bis-AAm) com N-hydroxyethylacrylamide (HEA) monómeros contendo um grupo hidroxilo primário formado por polimerização por radicais de uma rede aleatória hidrófila de poliacrilamida com grupos hidroxilo incorporados (hidroxi-PAAm) . Neste protocolo, um peso de 65 mg de HEA deve ser diluída num volume de 1 ml de HEPES. Sabendo-se que a densidade da HEA é aproximadamente igual a um, vamos supor que obtemos um volume de t...

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

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

Apresentamos um novo hidrogel de poliacrilamida, chamada hidroxi-PAAm, que permite uma ligação direta das proteínas da MEC com custo ou experiência mínima. A combinação de hidroxi-PAAm hidrogéis com impressão microcontact facilita o controlo independente das muitas pistas de o microambiente celular natural para estudar mechanostransduction celular.

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 ...

preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Research

JoVE Journal

Bioengineering

13.2K visualizações.  Université de Mons.  Apresentamos um novo hidrogel de poliacrilamida, chamada hidroxi-PAAm, que permite uma ligação direta das proteínas da MEC com custo ou experiência mínima. A combinação de hidroxi-PAAm hidrogéis com impressão microcontact facilita o controlo independente das muitas pistas de o microambiente celular natural para estudar mechanostransduction celular. 

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

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon Crescimento Stretch: O Mecanotransdução do Crescimento Neuronal

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Bioengenharia

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Hidrogéis alginato para Tridimensional Cultura Órgão de ovários e tubas uterinas

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Enriquecimento protéico de baixo peso molecular em filmes finos de sílica mesoporosa para a descoberta de biomarcadores

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

A Síntese de hidrogéis RGD-funcionalizado como uma ferramenta para aplicações terapêuticas

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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 ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Síntese de hidrogeles com propriedades anti-deposição como membranas de purificação de água

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

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) ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Uma plataforma lab-on-a-chip para estimular a mecanotransdução de osteócitos e analisar resultados funcionais da remodelagem óssea

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

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 ...