This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

1. Preparação de Soluções de Hidrogel Alíquotas e associadas <ol><li> Preparação da solução precursora de gel de poliacrilamida. <ol><li> Preparar solução precursora em gel de poliacrilamida através da mistura de acrilamida (A) (40% w / v, M r 71,08 g / mol), a bisacrilamida reticulador (B) (2% w / v, r M 154,17 g / mol), e de- água ionizada nos percentuais de volume especificados na Tabela 1. NOTA: Estas soluções podem ser preparadas em grandes lotes e guardadas a 4 ° C durante até vários meses. <ol><li> ATENÇÃO: A acrilamida é tóxico por inalação ou ingestão, particularmente, quando em forma de pó: assim, de preferência, utilizar 40% w solução / v para reduzir os riscos de toxicidade. Manusear somente enquanto vestindo roupas de proteção, como luvas, óculos de proteção e um jaleco. Guarde em um recipiente resistente à luz, bem fechado na geladeira (&lt;23 ° C, área bem ventilada). </li><li> CUIDADO: bis-acrilamida é tóxico por inalação ou ingestão, particularmente,quando na forma de pó: assim, utilizar de preferência 2% w solução / v para reduzir os riscos de toxicidade. Manusear somente enquanto vestindo roupas de proteção, como luvas, óculos de proteção e um jaleco. Guarde em um recipiente resistente à luz, bem fechado na geladeira (&lt;4 ° C, área bem ventilada). </li></ol></li></ol></li><li> Preparação e utilização de N -Sulfosuccinimidyl-6- (4&#39;-azido-2&#39;-nitrofenilamino) hexanoato (sulfo-SANPAH, M r 492,40 g / mol) alíquotas. CUIDADO: Sulfo- SANPAH causa irritação ocular grave; lidar com luvas e adequada para os olhos ou a protecção face. Armazenar a -20 ° C após o recebimento e antes de alíquotas. <ol><li> Para armazenar sulfo-SANPAH: dissolver Sulfo-SANPAH em dimethylsulfoxane (DMSO) a 50 mg / ml. Alíquota da solução estoque em tubos de centrífuga de 50 micro de 20 ul por tubo. Congelamento flash em gelo seco ou em azoto líquido (um passo opcional, mas preferido para preservar a máxima eficiência reticulador) e armazenar a -80 ° C. NOTA: As alíquotas can ser armazenados durante vários meses. </li><li> Para utilizar sulfo-SANPAH: descongelar o brevemente aliquota e diluída em 480 ul de água desionizada. Use imediatamente. Sulfo- SANPAH hidrolisa rapidamente em água; por isso tome cuidado para realizar todas as etapas acima rapidamente. </li></ol></li><li> Preparação de persulfato de amónio (Mr 228,18 g / mol) alíquotas. NOTA: persulfato de amónio causa dos olhos, pele e irritação das vias respiratórias. Use equipamento de proteção individual adequados ao manusear. Pega de persulfato de amónio em pó em um exaustor química. Pó Armazenar em local seco, bem ventilado. Uma vez aliquotas, a solução diluída pode ser utilizada na bancada de trabalho. <ol><li> Dissolve-se o persulfato de amónio em água desionizada para se obter uma concentração final de persulfato de amónio de 10% w / v. Alíquotas e armazenar a -20 ° C. Descongelar imediatamente antes da utilização. </li></ol></li><li> Preparação de colágeno tipo I solução. <ol><li> Preparar solução de colagénio a 0,2 mg / ml por diluição do stock assimlução em 1x tampão fosfato salino (PBS) de pH 7,4. Guarde em breve gelo ou usar imediatamente após a diluição. </li></ol></li></ol> 2. Preparação Hidrogel (Veja a Figura 1) <ol><li> Preparação das lâminas de vidro hidrofóbicas. <ol><li> Coloque algumas gotas de uma solução hidrofóbica sobre uma placa de vidro e usar papel de seda para se espalhar por toda a superfície. Deixe o ar seco e limpe com um lenço de papel novamente para uniformizar o revestimento hidrofóbico. NOTA: Loja solução hidrofóbica em um gabinete inflamável à temperatura ambiente. Use equipamento de proteção individual ao manusear. Trabalhar em uma área bem ventilada. </li></ol></li><li> Preparação do suporte de plástico flexível. <ol><li> Cortar o suporte de plástico flexível para corresponder ao tamanho da placa de vidro com revestimento hidrofóbico. </li><li> Marcar o lado hidrofóbico do suporte de plástico flexível, arranhando a superfície levemente com uma ferramenta afiada, como um bisturi. NOTA: Uma vez que o gel - que é totalmente transparente - Seca, As marcas de arranhões vai ajudar a distinguir de que lado o apoio de plástico flexível contém o gel. NOTA: O suporte de plástico flexível tem um lado hidrofóbico e um hidrofílico. Uma vez depositado, poliacrilamida adere permanentemente ao lado hidrofílica do suporte de plástico que facilita o manuseio fácil hidrogel subseqüente. </li></ol></li><li> Preparação de Gel (exemplo volumes são dados para 5 ml de soluções precursoras de gel). NOTA: 5 ml seria suficiente para produzir 40 ~ géis quando a espessura inicial de gel é de 0,5 mm. Mais géis podem ser produzidos, se a espessura do gel é reduzida. <ol><li> Colocar 4972,5 ul de solução precursora de poliacrilamida de concentração final desejada (Tabela 1) para um tubo de 50 ml. Colocar em uma câmara de desgaseificação durante 30 minutos, com a tampa aberta. NOTA: O oxigênio age como uma armadilha de radicais livres e, quando presente, ele inibe a polimerização. </li><li> Adicionar 25 ul de 10% w / v de persulfato de amónio (ver ponto 1.3) para alcançar a solução definitivapersulfato de amónio concentração de 0,05%. </li><li> Adicionar 2,5 mL de N, N, N &#39;, N&#39;-tetrametiletilenodiamina (TEMED, M r 116,24 g / mol) para a solução de gel desgaseificado para obter uma concentração de TEMED final de 0,5%. NOTA: Loja TEMED em um gabinete inflamável à temperatura ambiente. Manusear de um fume hood química quando vestindo equipamentos de proteção individual adequados. Manter hermeticamente fechada sob um gás inerte, uma vez que é altamente sensível à humidade e ar. </li><li> Misture a solução suavemente por pipetagem cima e para baixo 3-5 vezes. Não vórtice para evitar a difusão do oxigênio na solução precursor gel. </li><li> Pipeta a solução de gel para o lado hidrofílica do suporte de plástico flexível e sanduíche com hidrofóbica revestido lâmina de vidro. Separa-se as duas lâminas com espaçadores de silicone de espessura desejada (por exemplo, 0,5 mm). Deixar um pequena quantidade (~ 100 mL) de solução de precursor de polímero no tubo de 50 ml para usar como um indicador de gelificação. NOTA:Qualquer espaçador pode ser utilizado. Por exemplo, uma única tira de Parafilm dá uma espessura de gel inchado definitiva entre 100 - 120 um. </li><li> Coloque outra placa de vidro em cima do sanduíche apoio / gel de plástico flexível para averiguar uma superfície uniforme hidrogel após polimerização. </li><li> Deixe o gel polimerizar durante 45 minutos, embora menos tempo pode ser usado para géis de maior% em peso, se desejado. Para verificar se o gel tenha polimerizado, observar a solução restante no tubo de 50 ml; se a solução restante foi gelificada, então é provável que a gelificação na placa de vidro ter sido iniciado assim. No entanto, note que a abertura prematura do molde vai evitar a polimerização completa. </li><li> Uma vez formado o gel, retire o suporte de plástico flexível com o gel de poliacrilamida covalentemente ligado no topo, e do lado do gel de definir-se secar ao ar. NOTA: convecção de calor ou de secagem também podem ser utilizados para acelerar este passo. Depois secou-se sobre o suporte de plástico flexível, o gel pode ser lojad indefinidamente. <ol><li> Marcar algum ponto desencapado do suporte de plástico flexível, onde géis de poliacrilamida não formam devido a bolhas. Mark, enquanto o gel ainda é hidratado, pois isso vai misturar-se com o apoio de plástico flexível, uma vez que o gel é seco. </li></ol></li></ol></li></ol> 3. Assembléia Multiwell Plate, Collagen Coating, e Esterilização <ol><li> Conjunto da placa Multiwell. <ol><li> Uma vez seca, cortada géis PA nas formas desejadas. <ol><li> Para uma placa de 96 poços, usar um furador pesado com um diâmetro de ~ 6 mm. Use um cortador de papel pesados ​​para cortar géis em formas quadradas ou retangulares. Como alternativa, use uma tesoura. </li></ol></li><li> Prepare a cerca de 500 ul de PDMS por placa de 96 poços de acordo com as instruções do fabricante. Para os geles de cola para a parte inferior de uma placa com múltiplas cavidades, colocar uma pequena gota (~ 5 mL) de polidimetilsiloxano (PDMS) no centro de cada poço a. Utilizando uma pinça, coloque uma poliacrilamidae gel em cada lado de suporte de plástico bem, flexível para baixo. Para permitir que o PDMS para curar, deixar a placa montada a 37 ° C durante um mínimo de 4 h. </li></ol></li><li> Revestimento de colagénio de géis de poliacrilamida. <ol><li> Com uma pipeta de transferência, coloque uma pequena quantidade (7-8 ul) de sulfo-SANPAH (vide ponto 1.2.2) em cada poço e redemoinho de lado a lado para revestir a superfície do gel uniformemente. Trabalhe rapidamente desde sulfo-SANPAH não é estável em água. </li><li> Colocar a placa bem sob uma lâmpada de UV de alta intensidade (intensidade = 37 mW, λ = 302-365 nm) durante 5 min. Lavar os géis com PBS para remover o excesso de sulfo-SANPAH. </li><li> Pipetar 50 ul de 0,2 mg / ml de colagénio tipo I solução (ver ponto 1.4) em cada poço. Deixar a placa coberta à temperatura ambiente durante pelo menos 2 horas ou O / N a 4 ° C. NOTA: Para acelerar o processo de revestimento de colagénio, uma pipeta de transferência pode ser usado para adicionar a solução de colagénio. Tipicamente, 1-2 gotas da solução deve ser suficiente para completarly cobrir a superfície do gel. </li><li> Deixar a placa com cavidades à temperatura ambiente durante pelo menos 2 h, para permitir a ligação do colagénio. </li><li> Lavar com PBS para remover o excesso de solução de colagénio e esterilizar sob UV (λ = 200 nm) num capuz de cultura de tecido durante 2 horas. </li><li> Mergulhe os géis em meio completo (veja seção 4.1 para médias composição) O / N para hidratar e equilibrar. Use para celular semear imediatamente ou guarde na geladeira por até 2 dias. </li></ol></li></ol> 4. Sementeira celular no PA Rigidez Assay NOTA: Embora típico para linhas de células de mamíferos comuns, o protocolo apresentado nesta seção é usado especificamente com o câncer de mama linha de células MDA-MB-231 (ver Figuras 4 e 5). <ol><li> Recolher as células a partir de frascos de cultura de tecidos por exposição a 5% de tripsina / EDTA durante 5 minutos a 37 ° C. Use ~ 80 mL de tripsina / EDTA por cada cm 2 de área garrafa de cultura; por exemplo, utilizar 2 ml de tripsina / EDTA fora T25 frasco de cultura de células. Re-suspender as células recolhidas em meio completo suplementado com 10% de soro fetal bovino (FBS) e 1% de penicilina / estreptomicina a uma concentração celular final desejado. Tendo em conta o tempo de duplicação celular, bem como a duração do tempo as células serão semeadas no ensaio, escolher uma concentração de células sub-confluente apropriado. Certifique-se de que o meio adicionado é o suficiente para submergir completamente os hidrogéis. NOTA: Uma vez que os hidrogéis foram pré-equilibrada em mídia (consulte a etapa 3.2.6) 100 mL de volume deve ser suficiente. Os volumes médios típicos usados ​​para placas com múltiplas cavidades deve ser suficiente uma vez que os geles tenham sido pré-equilibrada em meios na etapa anterior. NOTA: O ensaio seria adequado para qualquer tipo de célula dependente de fixação. <ol><li> Contar o número de células em microscópio invertido usando um hemocitômetro. Carga de 10 ul de suspensão de células em cada porta de hemacitómetro e a média da contagem de células a partir de, pelo menos, 8 quadrantes. Para obter oconcentração celular final, multiplicar o número de células por 10 4. NOTA: Os melhores resultados de contagem de células são obtidos quando o número de células em cada quadrante hemacitï¿½etro é 20-50. </li></ol></li><li> Cultura das células para o ensaio de rigidez PA em um incubador humidificado a 37 ° C e 5% de CO 2. Mudança de mídia a cada 2-3 dias. Recolher as células, quando necessário por exposição a 5% de tripsina / EDTA a 37 ° C durante 5 min. Use ~ 80 mL de tripsina / EDTA por cm2 de garrafa de cultura de tecidos. Durante todas as etapas de manipulação celular, ter um cuidado especial para não perturbar a superfície do hidrogel: aspirado ou médio pipeta ligeiramente derrubando a placa com vários poços de lado e tocar a ponta da pipeta na parede lateral de cada bem, ao contrário de tocar o hidrogel. NOTA: Com excepção para tomar especial cuidado para não danificar a superfície do hidrogel durante a manipulação padrão de cultura de tecidos, as células semearam-se sobre o ensaio de rigidez podem ser manipulados da mesma maneira como se eles foram semeadas sobrepara uma placa com múltiplas cavidades regulares. </li></ol> 5. imagens de células semeadas em PA Rigidez Assay <ol><li> Células de imagem diretamente sobre o ensaio de rigidez PA. NOTA: Qualquer microscópio - invertido, fluorescente, ou confocal, pode ser usado para imagens de células. NOTA: O suporte de plástico flexível utilizado para construir o ensaio de rigidez é totalmente transparente e não autofluoresce ou interferir com imagens de células. No entanto, apesar de o próprio suporte de plástico flexível é transparente, capacidades de imagem será limitada pela distância de trabalho do objectivo. O suporte de plástico flexível tem uma espessura de 0,23 mm e uma distância de trabalho típico de uma objectiva 10X é ~ a 4 mm, para diminuir acentuadamente maiores ampliações. <ol><li> Para imagens de células vivas, a posição de ensaio de rigidez PA no suporte da placa de microscópio e imagem. Mantenha as sessões de imagem com menos de 2 horas, ou usar um microscópio equipado com uma câmara ambiental para tempos mais longos de imagem. </li><li> Quando estou de células vivasenvelhecimento não é o objetivo, corrigir as células em solução de formaldeído 4% suplementado com 0,1% de detergente. Soak células em fixador à temperatura ambiente durante um mínimo de 2 horas. Enxágüe com PBS duas vezes. Descarte de resíduos fixador em um recipiente de resíduos designada. NOTA: As células podem ser visualizados de imediato ou armazenados submersa em PBS a 4 ° C durante até 2 semanas. CUIDADO: O formaldeído é tóxico por inalação e contato. Manusear com luvas em uma coifa química. NOTA: protocolo de fixação celular tem que ser escolhido com base nos coloração celular e manipulação de protocolos posteriores, porque certos fixadores danificar algumas proteínas celulares. </li><li> No caso da imagiologia fluorescente, mancha células com coloração de células desejada directamente na placa com múltiplas cavidades fixo. Imagem imediatamente. </li></ol></li></ol>

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The authors have nothing to disclose.

Geles de poliacrilamida, originalmente desenvolvidos para electroforese, 28 são rotineiramente utilizados como substratos de cultura celular para estudar os efeitos de rigidez do substrato sobre a morfologia celular, motilidade, 3,24,29 e comunicação entre outras características celulares. Poliacrilamida permite a manipulação de rigidez do substrato para abranger a rigidez de todos os tecidos moles do corpo (0,3-300 kPa) 1 com uma simples mudança na concentração de precursor de ...

A maioria dos tecidos do corpo são materiais viscoelásticos moles com um módulo de Young na gama de 0,1 kPa para o cérebro a 100 kPa durante cartilagem mole, ainda, a maior parte da investigação de células in vitro é realizada em culturas de tecidos de poliestireno (TCP), que tem um módulo de ~ 1 GPa . 1 Esse descompasso rigidez afeta significativamente a forma como as células respondem ao seu meio ambiente. Um crescente corpo de pesquisa é, assim, dedicado a elucidar o efeito de rigidez do substrato sobre o destino de vários tipos de células, incluindo células estaminais 2,3 4 Como resultado., Vários hidrogéis têm sido desenvolvidos para auxiliar no entendimento de células dependente de rigidez biologia incluindo poliacrilamida (PA), 5-7 polietileno glicol (PEG), 8,9 polidimetilsiloxano (PDMS), 10 e 11 de alginato. Embora a evidência de que a rigidez substrato tem um impacto substancial sobre o destino celular está crescendo, a maioria dos estudos são realizados em uma pequena escala com um pequeno número de samples. Multidimensionais, estudos sistemáticos sobre o efeito da rigidez substrato para uma variedade de tipos de células ou condições ambientais são raros. 12 Várias tecnologias de hidrogel de alto rendimento promissoras foram desenvolvidas, incluindo microarranjos baseados em PEG, 13 dispositivos microfluídicos para a produção de microesferas de agarose de hidrogel, 14 ou micro e nano-hastes onde rigidez é modulada pelo diâmetro e altura das microrods. 15 No entanto , as tecnologias para preparar tais substratos são sofisticados e disponível para um número limitado de laboratórios. Muita pesquisa envolvendo rigidez modulado respostas das células utiliza poliacrilamida (PA) géis que não só são baratos e simples de implementar, mas também exibem uma gama fisiologicamente relevante de módulo de Young, a saber, 0,3 -. 300 kPa 16-22 No entanto, os métodos existentes para fabricar PA géis para cultura de células são intensivos de trabalho e, consequentemente, prepared em pequenos lotes. Algumas das dificuldades associadas com a preparação de géis PA como substratos de células-tronco a partir da exigência de que os géis tem que estar preparado: 1) na ausência de oxigênio para permitir a polimerização completa, 2) com uma superfície plana e lisa para permitir a célula uniforme ligação e de espalhamento, e 3) fixada permanentemente na parte inferior da placa de cultura de células para prevenir flutuação. Diversos grupos tentaram produzir géis PA para cultura de células em grandes lotes. Semler et al., Folhas de espessura preparados de géis de PA, que foram depois &quot;cortado&quot; com um furador e colocadas em placas de 96 poços. 23 No entanto, este método está limitado a géis mais duras, ou seja,&gt; 1 kPa no módulo de Young, porque mais suave géis são &quot;pegajoso&quot;, difícil de cortar, e facilmente danificada. Mih et ai. Desenvolveram uma técnica mais sofisticado que permite que os géis a ser polimerizado directamente em uma placa com múltiplas cavidades com fundo de vidro. <sup&gt; 6 Isto foi conseguido através de derramamento as soluções de gel em placas com fundo de vidro funcionalizados e formando géis por &quot;imprensando-los&quot; com uma matriz lamela personalizado. 6 embora muito promissores, efeitos ligeira vantagem ainda foram observados com esta técnica. Além disso, a técnica requer uma matriz de design personalizado não imediatamente acessível para muitos laboratórios, bem como placas com múltiplas cavidades com fundo de vidro caros. Este artigo descreve uma maneira simples e barata para montar géis PA em um prato com vários poços que poderiam ser facilmente adotado por qualquer laboratório. Aqui, um suporte de plástico flexível é utilizado, que tem dois lados - um um hidrofóbico, que é repelente para géis de PA, e um uma hidrofílica, que liga covalentemente o gel PA sobre deposição. Uma vez que as folhas de gel de PA são depositados e permanentemente fixo ao suporte de plástico flexível, que permite manipular géis de qualquer espessura ou rigidez e cortando-os em qualquer forma desejada. Este aproxoach não só produz personalizados lamelas &#39;&#39; plásticas em tamanhos não anteriormente disponíveis no mercado, mas também elimina a necessidade de superfícies de vidro-tratamento pré, quer lamelas de vidro ou os poços de placas de múltiplos poços com fundo de vidro caros, com uma solução de ligação PA, que é uma tediosa e um passo consumidor de tempo. Finalmente, as folhas de geles uniformes PA pode ser preparado em grandes lotes e armazenados de-hidratado durante vários meses. Em resumo, o ensaio aqui apresentado constitui uma melhoria em relação aos métodos existentes em vários aspectos. Em primeiro lugar, o processo de montagem de placa de poços múltiplos é eficaz, e o custo global dos materiais necessários é baixo. Em segundo lugar, os hidrogéis são produzidas em grandes lotes de um único filme em gel homogénea. Finalmente, apenas os materiais que se encontram comercialmente disponíveis são necessários. A utilidade do ensaio é ilustrada a explorar o efeito da rigidez do substrato na morfologia celular e a área de difusão.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Reagents</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">40% Acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">2% Bis-acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Ammonium Persulfate</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-07000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TEMED</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Sulfo-SANPAH</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:187px;">22589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Collagen Type 1, from Rat tail, 3.68 mg/mL</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:187px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:200px;">Fisher Scientific&#160;</td>
			<td style="width:187px;">BP231-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Hydrophobic solution - Repel Silane&#160;</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">17-1332-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">PBS (1x), pH 7.4</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:303px;">Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td style="width:200px;">Elsworth Adhesives</td>
			<td style="width:187px;">3097358-1004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tyrpsin/EDTA (10x)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">44174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RPMI-1640 Medium (1x)</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30027-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30073-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin Streptomycin</td>
			<td style="width:200px;">MP Biomedicals</td>
			<td style="width:187px;">1670046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent - Triton-X</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Formaldehyde 37% Solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">A2153</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">BSA-based cell adhesion blocking kit - ECM Cell Adhesion Array Kit</td>
			<td style="width:200px;">Chemicon International</td>
			<td style="width:187px;">ECM540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">flexible plastic support - GelBond PAG Film for Polyacrylamide Gels</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">309819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass Plates</td>
			<td style="width:200px;">Slumpys</td>
			<td style="width:187px;">GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 mL Conicals</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">3181345107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 mL Conicals</td>
			<td style="width:200px;">FALCON</td>
			<td style="width:187px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Micro centrifuge tubes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">96-well plate (flat bottom)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">12565501</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:303px;">Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Glass Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Tips (1-200uL, 101-1000uL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2707509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Plastic Standard Disposable Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:187px;">13-711-9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Parafilm</td>
			<td style="width:200px;">PARAFILM&#160;</td>
			<td style="width:187px;">PM992</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Powder Free Examination Gloves</td>
			<td style="width:200px;">Quest</td>
			<td style="width:187px;">92897</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td style="width:200px;">Grace Bio-Labs</td>
			<td style="width:187px;">JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Large/non-disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Light and Flourescent Microscope (Axiovert 200M)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">3820005619</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope Software</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV oven</td>
			<td style="width:200px;">UVITRON</td>
			<td style="width:187px;">UV1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum chamber/degasser</td>
			<td style="width:200px;">BelArt</td>
			<td style="width:187px;">999320237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum pump for degasser</td>
			<td style="width:200px;">KNF Lab</td>
			<td style="width:187px;">5097482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tissue Culture Hood</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-425-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Chemical Fume Hood</td>
			<td style="width:200px;">KEWAUNEE</td>
			<td style="width:187px;">99151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Inverted Microscope (Axiovert 25)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">663526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Incubator</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-8500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Aid</td>
			<td style="width:200px;">Drummond Scientific Co.</td>
			<td style="width:187px;">P-76864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemacytometer</td>
			<td style="width:200px;">Bright-Line</td>
			<td style="width:187px;">383684</td>
			<td></td>
		</tr>
	</tbody>
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simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Poliacrilamida (PA) hidrogéis são amplamente usadas para testar as respostas celulares dependentes de rigidez. 17,24 por mistura de várias concentrações de acrilamida (A) e bis-acrilamida (B) pode-se fazer géis PA que abrangem a gama de rigidez da maioria dos tecidos moles em o corpo - 0,3 -.. 300 módulo de kPa Jovem 1 No entanto, a preparação de géis de poliacrilamida é tedioso e demorado, muitas vezes limitando a sua utilidade em aplicações &quot;high-throughput&quot;, como para a se...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Simples Multiwell Rigidez Ensaio para o Estudo das respostas das células Rigidez dependentes base de poliacrilamida

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.5K visualizações.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Vídeo: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotípica Colágeno Ensaio I: Uma Plataforma maleável para Avaliar Comportamento celular em um contexto de 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Bioengenharia

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Baseados em células de Ensaio de cálcio para Médio a High Throughput Screening de Funções TRP usando FlexStation Canal 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Ensaio de aderência de freqüência para In Situ Análise Cinética de Cross-juncional interações moleculares na interface célula-célula

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Aspiração micropipeta de Células-substrato anexados para Estimar Rigidez celular

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microinjeção Ferida Ensaio e<em&gt; In vivo</em&gt; Localização de Epidermal Wound resposta Jornalistas em<em&gt; Drosophila</em&gt; Os embriões.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

O uso de chips microfluídicos para Imagens ao vivo e estudo das respostas Lesão em<em&gt; Drosophila</em&gt; Larvas

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Análise 3D de respostas multicelulares a gradientes Quimioatrativos

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Ensaio padronizado e escalável para estudar perfundido 3D Angiogênico Sprouting de células endotelias derivadas do iPSC in vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.