Os autores agradecem W. Sun e K. Jansen para as discussões críticas, e reconhecer o apoio pelo Nano Laboratório de Biomecânica da Universidade Nacional de Cingapura. NAK reconhece o apoio por um Marie Curie IIF Fellowship.

Colheita 1. celular <ol><li> Obter MD-MBA-231 a partir do 37 ° C, 5% de CO2. Separar as células do tecido placa de cultura utilizando-se 0,5% de solução de Tripsina-EDTA. Use 1 ml de solução de tripsina-EDTA para células cultivadas num frasco T25. </li><li> Células de pellets em um tubo de 15 ml por centrifugação a 200 x g durante 4 min, aspirar o sobrenadante e ressuspender as células em 5 ml de meio de cultura. </li><li> Contagem de densidade celular, ρ, utilizando um hemocitómetro. Nota: Para preparar o gel interior da célula-semeado, a suspensão de células vai ser, mais tarde, diluiu-se 10 × para atingir a densidade final de células de semeadura. Portanto, 10 × suspensão concentrada de célula é necessário. </li><li> Calcula-se o volume de meio necessário para atingir uma concentração de 10 × celular: <img alt="Equação 1" fo:content-width="2in" src="/files/ftp_upload/52735/52735eq1.jpg" width="175" /> Nota: A densidade final semeadura de células, c final, de cerca de 215; 10 6 células / ml é recomendado para MD-MBA-231 e é utilizado no presente protocolo. Outros densidades de sementeira podem também ser explorado para outros tipos de células. </li><li> Células de pellets mais uma vez em um tubo de 15 ml por centrifugação a 200 × g por 4 min, e aspirar o sobrenadante. </li><li> Ressuspender as células na quantidade necessária (V médio calculadas no passo 1.3) de meio de cultura celular livre de soro cuidadosamente para minimizar a aglutinação celular. Nota: O fenol vermelho é auto-fluorescente, e pode interferir com a imagiologia por fluorescência / reflectância. O uso de meio isento de vermelho de fenol pode ser considerado para alcançar melhor qualidade de imagem. </li></ol> 2. Preparação de Soluções de colágeno <ol><li> Obter a solução de colagénio de estoque, 10 × tampão PBS, Milli-Q H 2 O, NaOH a 0,1 M, e vários tubos de microcentrífuga. Mantenha tudo no gelo para evitar a polimerização prematura de colágeno, e manter a condição estéril. </li><li> Equilibrar uma Glass- estérilprato inferior por pré-aquecendo-a no 37 ° C incubadora. Nota: Todos os volumes neste protocolo foram otimizados para um prato fundo de vidro de 12 mm assim. Se outro tipo prato é usado, ajustar os volumes proporcionalmente. </li><li> Calcular o volume requerido necessário para preparar 50 ul de solução de colagénio a 2,4 mg / ml para o gel interior (solução I) com base na concentração de estoque de colagénio. Nota: Outras concentrações de colagénio para o interior de gel pode também ser usado. </li><li> Em um ambiente estéril (tipicamente um capuz biosegurança), adiciona-se lentamente 5 mL de 10 × tampão PBS para a quantidade necessária de solução-mãe de colagénio (calculado no passo 2.3), com agitação suave. Tome cuidado para evitar a formação de bolhas de ar. </li><li> Ajustar o pH da mistura para 7,4 utilizando NaOH 0,1 M calibrado usando medidor de pH. Como orientação geral, utilizar cerca de 5 ul para trazer o pH para perto de 7,4 (a quantidade varia dependendo da concentração estoque e pH). Nota: Observe que os volumes envolvidos nestepasso é demasiado pequeno para o uso do medidor de pH padrão. Use um dos seguintes truques: <ol><li> Preparar soluções de colágeno para várias amostras. Ajustar o pH em grandes quantidades usando um medidor de pH padrão e distribuir as soluções de colagénio entre as amostras. </li><li> Em alternativa, ajustar o pH de uma solução de colagénio em maior volume (i. E., O volume que permitem o uso de medidor de pH padrão). Note-se a quantidade de NaOH necessária para trazer o pH para o valor final de pH. Reduza os volumes e usar a quantidade apropriada de NaOH para o experimento. Confirme o valor pH utilizando papel Litmus. </li><li> Caso contrário, utilizar um eléctrodo de pH de micro para ajustar de forma mais precisa o pH de pequenas quantidades. </li></ol></li><li> Levar a solução para um volume de 45 ul usando H 2 O. Execute todas as etapas no gelo para evitar a polimerização prematura de colágeno. </li></ol> 3. Formação de Concentric Cultura Gel <ol><li> Pegue o prato com fundo de vidro pré-aquecido (veja o passo 2.2) a partir de the incubadora. </li><li> Adiciona-se 5 ul da suspensão de células de 10 x concentrado (preparado no passo 1.5) a solução I. Ressuspender completamente. Tome cuidado para evitar a formação de bolhas de ar. A mistura agora tem um volume de 50 ul e contém a concentração final de colagénio (2,4 mg / ml) e a densidade celular (final c = 2 × 10 6 células / ml). </li><li> Adicionar 20 ul da solução contendo as células que se lentamente para o centro do poço, de modo a formar uma gotícula (Figura 1A) em forma de cúpula. Tome cuidado para evitar a formação de bolhas nesta etapa. Caso se forme um bolha, com cuidado, mas rapidamente quer tentar rompê-lo ou sugá-lo para fora usando uma ponteira. Suavemente colocar o prato de volta na incubadora para permitir que o gel interior polimerizar durante 45 minutos. </li><li> Preparar a solução de O (para o, gel de colagénio acelular exterior) cerca de 15 minutos antes do final deste passo de incubação. Nota: A condição exterior de gel pode ser variada em termos de concentração de colagénio e ao pH de polimerizaçãoobter redes com diferentes microestruturas 10. Neste protocolo, o foco sobre um 1,5 - gel de colagénio a 4,0 mg / ml polimerizada a um pH de 7,4. <ol><li> Com base na concentração de estoque de colagénio, calcular o volume requerido necessário para preparar 200 mL de solução de colagénio O na concentração final. </li></ol></li><li> Adicionar 20 ul de tampão PBS 10 × lentamente para a quantidade necessária de solução-mãe de colagénio (calculado no passo 3.4), com agitação suave. Ajustar o pH da mistura para o valor final de pH utilizando NaOH 0,1 M e o uso de medidor de pH calibrado. Veja a nota para o passo 2.5 relativamente ao ajuste de pH. </li><li> Levar a solução para um volume final de 200 ul usando H 2 O. Execute todas as etapas no gelo para evitar a polimerização prematura de colágeno. </li><li> Leve o prato da incubadora após 45 min de polimerização do gel interior (veja o passo 3.3). Adicionar cuidadosamente 180 ul de solução de S no topo do gel interior, de modo que a solução de cobre completamente o interiorgel e enche-se a cavidade (Figura 1B). <ol><li> Executar este passo cuidadosamente sem agitar a solução, o que pode levar a orientações de fibras não-uniformes no exterior de gel. Tome cuidado para evitar tocar o gel interno com uma ponteira, e para evitar a formação de bolhas ou bolsas de ar. Caso se forme um bolha, com cuidado, mas rapidamente quer tentar rompê-lo ou sugá-lo para fora usando uma ponteira. Suavemente colocar o prato de volta na incubadora para permitir que a polimerizar exterior de gel. </li></ol></li><li> Leve o prato da incubadora após 45 min de polimerização. O gel deve ser já bastante solidificado, neste ponto, embora ainda pode destacar da superfície inferior, se manuseada com cuidado. <ol><li> Suavemente para 2 ml de meio de cultura celular aquecida ao prato (Figura 1C). Certifique-se de que o gel é completamente submergido no meio. Atualize a médio a cada 2-3 dias ao longo do período de cultura. </li></ol></li></ol> 4. Imagens ao vivo de células <ol><li> Realizar imagem usando um microscópio confocal invertido equipado com capacidade de geração de imagens de células vivas de longo prazo. Incluir um embutido câmara de incubação com uma temperatura (37 ° C) e CO2 (5%) de controlo. Ligue o microscópio e aquecer o palco, pelo menos, 1 hora antes do início do experimento. Nota: Use lente objetiva com longa distância de trabalho para otimizar a observação e localização das células nos géis 3D. </li><li> Incubar o gel em meio contendo 5 ul de corante de rastreador de células fluorescentes durante 30 min para permitir uma localização precisa das células no sistema 3D. Posteriormente, remover o corante não ligado por lavagem três vezes com 1 × PBS. Em seguida, adicionar meio de cultura de células para o prato. </li><li> Leve o prato da incubadora e colocá-lo no palco microscópio (Figura 1D). Nota: imagens de células vivas pode começar em princípio, logo após a polimerização do gel exterior. No entanto, neste momento, as células in O gel interno não se espalharam ainda. Para examinar a migração das primeiras células que invadiram o exterior de gel, de imagem de células vivas pode começar 24 horas (dependendo do tipo de células) após o inicio da cultura. Como um guia, em torno de 12 - são necessárias mais 14 dias de cultura para a maioria das células no interior do gel para entrar no exterior de gel. </li><li> Selecionar volumes de Vista (VOV de) em regiões do gel exterior em torno do gel interior. Após 24 h de incubação, a população de células terá difundido, atravessou a interface entre os geles de interior e exterior, e começou a invadir o exterior de gel. <ol><li> Para o VOV de, incluem as regiões de gel imediatamente ao lado da interface de gel, regiões intermédias, e as regiões perto da periferia da gel exterior 7. Excluir regiões mais próximas do que 50 mm a partir das superfícies de fundo e laterais, bem como a partir do topo do gel, a fim de evitar possíveis efeitos de borda. Cada VOV tipicamente mede 647 × 647 × 100 mm 3 (em x, y, z e indicações, respectivamente), com intervalo de 5 mm na -stack z. </li></ol></li><li> Assegurar modos de imagem, canais / filtros, tempos de exposição, e resoluções de imagem estão corretamente selecionado. Para imagens de livre-label da rede de colágeno, utilize microscopia confocal de reflectância simultaneamente durante o lapso de tempo de imagens ao vivo de células. </li><li> Tome imagens de exemplo, traçar os histogramas de intensidade, e ajustar os ganhos e compensações para observar sinal suficiente e evitar a saturação, assegurando que o histograma fica entre zero e a intensidade máxima. Não altere estas configurações mais durante todo o período do experimento. </li><li> Tome imagens de lapso de tempo dos VOV de selecionados, com um intervalo de tempo, Δ t, de 10 min para 8 horas (ou mais, se necessário). </li></ol> 5. Acompanhamento de celular e Análise de Dados <ol><li> Realizar imagem quantitativa pós-processamento das imagens -stack z utilizando appropria<html> software de processamento de imagem te. <ol><li> Segmento as imagens de lapso de tempo para selecionar automaticamente as posições celulares 3D em (x, y, z, t). </li><li> Para cada quadro, verificar manualmente a precisão de localização e remover falsos positivos devido a restos de células e saliências celulares que podem ter sido confundido com células. Remover as células de proliferação a partir da análise e as células que se sobrepõem ou ligados divididas em objetos distintos. </li></ol></li><li> Gerar faixas de células de lapso de tempo em 3D a partir de células (as coordenadas x, y, z, t) obtidos no passo anterior, ligando a localização de cada célula na sequência de tempo. </li><li> Elimine o ruído aleatório e sistema removendo faixas mais curtos do que um comprimento de rasto limiar (tipicamente 20 minutos). </li><li> Corrija para a amostra deriva subtraindo os deslocamentos líquidos globais dos trilhos, se necessário. </li><li> Calcular o deslocamento de células,iles / ftp_upload / 52735 / 52735eq2.jpg &quot;width =&quot; 80 &quot;/&gt;, e distância de migração celular, <img alt="Equação 3" fo:content-width="1in" src="/files/ftp_upload/52735/52735eq3.jpg" width="100" /> , A partir de células observadas as trajectórias, em que é um vector que representa a localização de uma célula de 3D em tempo e é o número total de pontos de tempo. <ol><li> Calcular a velocidade célula como S = d / (n • Δ t), onde Δ t é o intervalo de tempo entre os quadros. Calcule direcionalidade migração celular (ou persistência) usando P = Δd / d. Esta simples medida de persistência implica que, para P = 0 o deslocamento líquido é zero e para P = 1 a trajetória é uma linha reta direcional. </li></ol></li></ol>

<ol>
	<li>Horwitz, R., Webb, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration.">Cell migration.</a> Curr Biol. 13 (19), R756-R759 (2003).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat. Protoc. 2 (2), 329-333 (2007).</li><li>Provenzano, P. P., Eliceiri, K. W., Inman, D. R., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+three-dimensional+collagen+matrices+to+provide+contact+guidance+during+3D+cell+migration.">Engineering three-dimensional collagen matrices to provide contact guidance during 3D cell migration.</a> Curr. Prot. Cell Biol. 10, 10-17 (2010).</li><li>Friedl, P., Sahai, E., Weiss, S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+dimensions+in+cell+migration.">New dimensions in cell migration.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 743-747 (2012).</li><li>Grinnell, F., Petroll, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+and+mechanics+in+three-dimensional+collagen+matrices.">Cell motility and mechanics in three-dimensional collagen matrices.</a> Annu. Rev. Cell. Dev. Biol. 26, 335-361 (2010).</li><li>Wirtz, D., Konstantopoulos, K., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physics+of+cancer:+the+role+of+physical+interactions+and+mechanical+forces+in+metastasis.">The physics of cancer: the role of physical interactions and mechanical forces in metastasis.</a> Nat. Rev. Cancer. 11 (7), 512-522 (2011).</li><li>Sun, W., Kurniawan, N. A., Kumar, A. P., Rajagopalan, R., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+migrating+cell-induced+matrix+reorganization+on+3D+cancer+cell+migration.">Effects of migrating cell-induced matrix reorganization on 3D cancer cell migration.</a> Cell. Mol. Bioeng. 7 (2), 205-217 (2014).</li><li>Achilli, M., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+mechanical+properties+of+collagen-based+scaffolds+for+vascular+tissue+engineering:+the+effects+of+pH,+temperature+and+ionic+strength+on+gelation.">Tailoring mechanical properties of collagen-based scaffolds for vascular tissue engineering: the effects of pH, temperature and ionic strength on gelation.</a> Polymers. 2 (4), 664-680 (2010).</li><li>Kurniawan, N. A., Wong, L. H., Rajagopalan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stiffening+and+softening+of+collagen:+interplay+of+deformation+mechanisms+in+biopolymer+networks.">Early stiffening and softening of collagen: interplay of deformation mechanisms in biopolymer networks.</a> Biomacromolecules. 13 (3), 691-698 (2012).</li><li>Sun, W., Lim, C. T., Kurniawan, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+adaptability+of+cancer+cells+strongly+affects+anti-migratory+drug+efficacy.">Mechanistic adaptability of cancer cells strongly affects anti-migratory drug efficacy.</a> J. R. Soc. Interface. 11 (99), 20140638 (2014).</li><li>Guzman, A., Ziperstein, M. J., Kaufman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+fibrillar+matrix+architecture+on+tumor+cell+invasion+of+physically+challenging+environments.">The effect of fibrillar matrix architecture on tumor cell invasion of physically challenging environments.</a> Biomaterials. 35 (25), 6954-6963 (2014).</li><li>Wolf, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+limits+of+cell+migration:+control+by+ECM+space+and+nuclear+deformation+and+tuning+by+proteolysis+and+traction+force.">Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force.</a> J. Cell Biol. 201 (7), 1069-1084 (2013).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat. Rev. Mol. Cell Biol. 10 (7), 445-457 (2009).</li><li>Vedula, S. R. K., Ravasio, A., Lim, C. T., Ladoux, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration:+a+mechanistic+perspective.">Collective cell migration: a mechanistic perspective.</a> Physiology. 28 (6), 370-379 (2013).</li><li>Petrie, R. J., Doyle, A. D., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat. Rev. Mol. Cell Biol. 10 (8), 538-549 (2009).</li><li>Miron-Mendoza, M., Seemann, J., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+regulation+of+cell+motile+activity+through+matrix+stiffness+and+porosity+in+three+dimensional+collagen+matrices.">The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices.</a> Biomaterials. 31 (25), 6425-6435 (2010).</li><li>Mouw, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+mechanics+modulate+microRNA-dependent+PTEN+expression+to+regulate+malignant+progression.">Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression.</a> Nat. Med. 20 (4), 360-367 (2014).</li><li>Wolf, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-based+cell+migration+models+in+vitro+and+in+vivo.">Collagen-based cell migration models in vitro and in vivo.</a> Semin. Cell Dev. Biol. 20 (8), 931-941 (2009).</li><li>Wong, L. H., Kurniawan, N. A., Too, H. -. P., Rajagopalan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+resolved+microrheology+of+heterogeneous+biopolymer+hydrogels+using+covalently+bound+microspheres.">Spatially resolved microrheology of heterogeneous biopolymer hydrogels using covalently bound microspheres.</a> Biomech. Model. Mechanobiol. 13 (4), 839-849 (2014).</li><li>Gupton, S. L., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+feedback+between+actomyosin+and+focal-adhesion+systems+optimizes+rapid+cell+migration.">Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.</a> Cell. 125 (7), 1361-1374 (2006).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+guidance+mediated+three-dimensional+cell+migration+is+regulated+by+Rho/ROCK-dependent+matrix+reorganization.">Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization.</a> Biophys. J. 95 (11), 5374-5384 (2008).</li><li>Wolf, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell Biol. 9 (8), 893-904 (2007).</li><li>Jansen, K. A., Bacabac, R. G., Piechocka, I. K., Koenderink, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+actively+stiffen+fibrin+networks+by+generating+contractile+stress.">Cells actively stiffen fibrin networks by generating contractile stress.</a> Biophys. J. 105 (10), 2240-2251 (2013).</li></ol>

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In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been lar...

A migração de células desempenha um papel chave em várias respostas fisiológicas, tais como o desenvolvimento embrionário, hemostase, e resposta imunitária, bem como em processos patológicos tais como doenças vasculares, inflamação e cancro 1. Dissecando os factores bioquímicos e biofísicos subjacentes a migração celular é portanto fundamentalmente importante não só para compreender os princípios básicos de funções celulares, mas também para fazer avançar várias aplicações biomédicas, tais como em engenharia de tecidos, anti-metástases e desenvolvimento de drogas anti-inflamatórias. Desde observação in vivo é tecnicamente desafiador, uma série de esforços tem sido focada em in vitro recapitulação da migração celular. Os métodos in vitro para estudar a migração de células têm sido amplamente concebido para ensaios em duas superfícies (2D) dimensionais, mais notavelmente o arranhão ou ferimento ensaio 2 de cura. Tais ensaios oferecem configuração experimental simples, fácil live-imagens de células, e fornecer informações úteis sobre vários mecanismos bioquímicos subjacentes migração celular. No entanto, estes ensaios não têm em conta a arquitetura da matriz extracelular (ECM) e remodelação, que são aspectos críticos na compreensão da migração vivo. Recentemente, tem sido cada vez mais apreciado que um modelo de cultura 3D, muitas vezes em matrizes à base de colagénio 3, fornece uma plataforma que melhor se assemelha à situação in vivo. De fato, as células apresentam dinâmicas migratórias que são distintas daquelas em superfícies 2D, especialmente devido à diferente dimensionalidade do meio ambiente 4. Além disso, as propriedades biofísicas e mecânicas da matriz afecta sensivelmente a migração celular 5, nomeadamente no contexto de invasão de células tumorais 6. Aqui, apresentamos um método para estudar o comportamento de migração celular 3D em ECM com propriedades biofísicas que podem ser facilmente variado, com condições de preparação. As células sãosemeadas em um &quot;gel interior&quot; e estão autorizados a escapar para dentro e invadir o &quot;gel exterior&quot; inicialmente acelular. O método baseia-se na capacidade da célula para reconhecer a presença de, e a propensão para migrar para dentro, regiões livres de células no exterior de gel, que está intimamente ligado à célula mechanosensing 7. Neste estudo, utilizamos redes de colágeno que a ECMs invadidas por células de câncer de mama altamente invasivos, MD-MBA-231. As propriedades mecânicas e microestrutura de ambos os géis de interiores e exteriores pode ser sintonizado 8 e 9 caracterizado para atingir as condições f isiologicamente relevantes. Reconstrução e análise das faixas de células permitir o exame quantitativa detalhada do comportamento de migração espaço-temporais, tanto a nível da população e nível de célula individual. É importante notar que a configuração do sistema de gel concêntrico imita a topologia de tecido in vivo enfrentado pelas células migrantes, especialmente células cancerosas invasoras, oferecendo, assim, conhecimentos importantespara os mecanismos físicos de migração celular e metástase.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture incubator</td>
			<td>Fisher Scientific Pte Ltd</td>
			<td>Model: 371, S/No 318854-6055</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon A1R</td>
			<td></td>
			<td>Inverted confocal laser scanning microscope equipped with incubator chamber</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent CellTracker dye CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom dish</td>
			<td>IWAKI Cell Biology</td>
			<td>3931-035</td>
			<td>35 mm diameter dish with 12 mm diameter glass-bottom well</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>iN CYTO</td>
			<td>DHC-N01 (Neubauer Improved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprocessor pH meter</td>
			<td>Hanna Instruments</td>
			<td>pH 211</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutragen Collagen</td>
			<td>Advanced BioMatrix</td>
			<td>#5010-D</td>
			<td>Acid-solubilized bovine collagen type I (stock pH ~ 2)</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sartorius</td>
			<td>S/No 29153352</td>
			<td>Basic pH Meter PB-11</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
	</tbody>
</table>

concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration

A capacidade das células para migrar é crucial numa grande variedade de funções celulares ao longo da vida do desenvolvimento embrionário e na cicatrização de tumor e a metástase do cancro. Apesar dos esforços intensos de investigação, os princípios bioquímicos e biofísicos básicos de migração de células ainda não são totalmente compreendidos, especialmente nas três dimensões (3D) microambientes fisiologicamente relevantes. Aqui, nós descrevemos um ensaio in vitro concebido para permitir a análise quantitativa de 3D ​​comportamentos de migração celular. O método explora mechanosensing capacidade da célula e propensão para migrar para dentro da matriz extracelular previamente desocupado (ECM). Usamos a invasão de células do cancro da mama altamente invasivos, MDA-MB-231, em géis de colagénio, tal como um sistema modelo. A disseminação da população de células e as dinâmicas migratórias de células individuais ao longo de semanas de cultura pode ser controlada por meio de imagens ao vivo de células e analisados ​​para extrair dados espaço-temporalmente-resolvidos. Além Do Mais, O método é facilmente adaptável para diversas matrizes extracelulares, oferecendo assim uma maneira simples e poderosa para investigar o papel dos factores biofísicos no microambiente sobre a migração celular.

O ensaio de gel concêntrico aqui apresentada foi realizada utilizando células de cancro da mama altamente invasivos, MDA-MB-231, com 2,4 mg / ml de gel de colagénio interior e uma densidade de sementeira de células de = 2 x 10 6 células / ml, como um exemplo. Como mostrado na Figura 2, tipicamente depois de alguns dias de cultura, as células violou a interface gel interior-exterior e começou a invadir o exterior de gel. A população de células predominantemente espalhar radialmente p...

Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

As propriedades mecânicas e a micro estrutura da matriz extracelular afectar fortemente a migração de células 3D. Um método in vitro para estudar o comportamento de migração celular no espaço-temporal ambientes biofísico variáveis, tanto na população e os níveis de células individuais, é descrito.

Sistema de Gel Concentric para estudar o papel Biofísica da Matrix Microenvironment sobre a migração celular 3D

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

concentric-gel-system-to-study-biophysical-role-matrix

Research

JoVE Journal

Bioengineering

8.5K visualizações.  FOM Institute AMOLF.  As propriedades mecânicas e a micro estrutura da matriz extracelular afectar fortemente a migração de células 3D. Um método in vitro para estudar o comportamento de migração celular no espaço-temporal ambientes biofísico variáveis, tanto na população e os níveis de células individuais, é descrito. 

Vídeo: Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Bioengenharia

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Estudo da migração celular em Canais microfabricados

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Gradiente planar do sistema de difusão para investigar Quimiotaxia numa Matriz de Colagénio 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

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

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

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

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

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

Um modelo de cultura de matriz-adipócito extracelular humano para estudar crosstalk metabólico de matriz e células

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

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Rotulagem de vesículas extracelulares para monitoramento de migração e absorção em Explants de cartilagem

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

Suporte composto de matriz extracelular microgel para impressão 3D incorporada de construções neurais humanas

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...