O financiamento foi fornecido pelo National Institutes of Health (EB 001.013), Ciências Naturais e Pesquisa de Engenharia Council of Canada e Canadian Institutes of Health Research. Agradecemos ao Dr. AP McGuigan por sua experiência e ajudar no desenvolvimento de módulos.

1) Preparação de Tubos <ol><li> Corte 2-3 comprimentos m de tubos de polietileno (0,76 mm de diâmetro x 1,22 milímetros OD). </li><li> Um fio de agulha 20 G1 em uma das extremidades do tubo. </li><li> Tubos de bobina e coloque em uma bolsa de conversores auto-vedação (7 1 / 2 &quot;x 13&quot;). As duas extremidades do tubo devem ser colocados no final unsealed do saco e gravado no local com fita de esterilização a gás. </li><li> Saco de vedação e gás esterilizar. </li></ol> 2 Neutralização) de colágeno Nota: 1 ml de colágeno preenche cerca de 2 m de tubos de polietileno. <ol><li> Coloque a quantidade necessária de colágeno necessário para o comprimento desejado de tubo em um tubo cônico de 15 ml e manter em gelo. </li><li> Adicionar 128 uL de 10x α-MEM por mL de colágeno para o tubo e misturar por pipetagens repetidas da solução. Nota: (1) A solução deve girar o amarelo como o α-MEM é misturado na colágeno acidificado e (2) tentar minimizar bolhas, quando a mistura. </li><li> Neutralizar o colágeno, adicionando bicarbonato de sódio 0,8 M até que o colágeno tem um pH de 7,4. Para uma estimativa geral do bicarbonato de sódio pH adicionar até a solução de colágeno amarela vira uma cor rosa claro. Nota: Ele normalmente leva 30-60 mL por cada mL de colágeno. </li><li> Mantenha colágeno neutralizado em gelo até estar pronta para usar como o colágeno neutralizado vai gel se não forem mantidas a 4 ° C. </li></ol> 3) Incorporação de Cells (Opcional) Nota: Dependendo do tipo de célula que as células podem ser incorporados em uma concentração de entre 1 milhão de células por ml a 20 milhões de células por mL. Use o meio necessário para as células que você deseja incorporar. <ol><li> Remova a mídia a partir de células e lavar com 5 mL de PBS. </li><li> Remover PBS e adicionar 3 mL de tripsina-EDTA. </li><li> Incubar as células em tripsina-EDTA por 5 min a 37 ° C. </li><li> Ressuspender as células em 7 mL de mídia. </li><li> Contagem de células. </li><li> Transferir o número necessário de células para a incorporação de um tubo cônico de 15 mL. </li><li> Agregar as células a 300 xg por 5 min. </li><li> Aspirar mídia fora do pellet de células. </li><li> Ressuspender as células do colágeno neutralizada e manter em gelo. </li></ol> 4) gelificação do Colágeno <ol><li> Em um armário de segurança biológica corte a extremidade selada do saco que contém o tubo de polietileno. </li><li> Anexar uma seringa de 3 ml para a agulha 20G, no final, um dos tubos, usando uma pinça esterilizada. Manter o tubo no saco. </li><li> Insira a outra extremidade do tubo no colágeno neutralizado até que a ponta está no fundo do tubo cônico, usando uma pinça esterilizada. Manter o tubo cônico no gelo. </li><li> Puxe o colágeno na tubulação de polietileno retraindo o êmbolo da seringa devagar. Nota: Se o colágeno é puxado para dentro do tubo de forma rápida, em seguida, irá formar bolhas no colágeno. </li><li> Depois de puxar o colágeno através do tubo, puxe a agulha 20G de tubulação e colocar ambas as extremidades de volta para o saco. Saco de fita fechada. </li><li> Incubar a bolsa a 37 ° C por 60 min. Se o colágeno contém células, então, virar o saco sobre cada 15 minutos. para que as células não se contentar com um lado do módulo. </li></ol> 5) Corte de Tubos Contendo o colágeno coagulada <ol><li> Cabeçalhos autoclave e lâmina para o dispositivo de corte e bandeja para segurar a tubulação. </li><li> Montar o dispositivo de corte. </li><li> Tubulação lugar em uma bandeja estéril na frente do dispositivo de corte e carga do tubo no dispositivo de corte. </li><li> Configurar um tubo cônico de 50 mL contendo 25 mL dos meios de comunicação na saída do dispositivo de corte para recolher os módulos de corte. Nota: Use mídia contendo FBS mesmo para colágeno somente módulos como os módulos não se separam da tubulação sem FBS na mídia. Se os módulos contêm células usam os meios de comunicação necessários para as células incorporado. Se não houver células incorporados nos módulos usar a mídia para as células endoteliais. </li><li> Definir o dispositivo de corte para cortar 2 mm de comprimento tubos e cortar a tubulação. </li><li> Incubar módulos corte a 37 ° C por 60 min no tubo cônico de 50 mL. </li></ol> 6) A remoção do colágeno Módulos de Tubulação <ol><li> Vortex tubo cônico de 50 mL contendo módulos na tubulação por 10 seg. Nota: Seja gentil quando separar módulos integrados com alta densidade de células para evitar a ruptura. </li><li> Deixe módulos para liquidar o fundo do tubo. Leva cerca de 5 min. </li><li> Usando uma boca larga pipeta 10 mL sorológicos transferir os módulos resolvido a um tubo de 15 mL cônico. </li><li> Repita os passos 6,1-6,3 duas vezes. </li><li> Vá para a etapa 7 para módulos de revestimento com células endoteliais ou módulos de transferência para uma placa de cultura não-tecido tratado. </li></ol> 7) Revestimento os módulos com células endoteliais <ol><li> Settle módulos no fundo de um tubo cônico com 15 mL 5 mL dos meios de comunicação para poe incorporados células. </li><li> Remova a mídia a partir de células endoteliais e lavar com 5 mL de PBS. </li><li> Remover PBS e adicionar 3 mL de tripsina-EDTA. </li><li> Incubar as células em tripsina-EDTA por 5 min a 37 ° C. </li><li> Ressuspender as células em 7 mL de mídia. </li><li> Contagem de células. Precisa de 5x10 6 células endoteliais por mL de módulos embalado na parte inferior do tubo cônico. </li><li> Agregar as células a 300 xg por 5 min e ressuspender em 5 mL de mídia das células endoteliais. </li><li> Adicione o de 5 mL da mídia contendo células endoteliais para os módulos. Nota: Isto dá uma proporção de 50:50 de dois tipos de mídia que nós encontramos para trabalhar para a sobrevivência ea função de ambos os tipos de células. Testar o uso de um meio de co-cultura formulada para garantir a manutenção adequada dos fenótipos dos dois tipos de células antes de usá-lo. </li><li> Incubar os módulos com as células endoteliais tanto estática e dinamicamente. </li><li> Para os módulos de incubação estática misturar com células endoteliais por inversão e definir o tubo na posição vertical a 37 ° C por 15 min. Repetir. </li><li> Dinamicamente a semente células endoteliais-o suavemente o tubo a 37 ° C por 60 min. </li><li> Repita duas vezes de incubação estática. </li><li> Coloque os módulos em uma placa de cultura de tecidos não-tratados e incubar durante a 37 ° C. </li><li> O lugar no dia seguinte os módulos em uma placa nova cultura não-tecido tratado com meios frescos (mistura 50:50, se usar o sistema de co-cultura) para remover unattached células endoteliais. </li><li> Incubar módulos que são revestidos com células endoteliais até cobrir 100% da superfície dos módulos. Isso geralmente leva cerca de 7 dias. </li></ol> 8) Resultados Representante: Os módulos vão olhar cilíndrica quando eles são removidos do tubo. Se os módulos contêm células incorporados e / ou são revestidos com células endoteliais que podem contratar até 50% em volume e desenvolver uma forma oval (Figura 1). Os módulos também se tornará mais densa e mais opaco quando vistos por microscopia de luz (Figura 1). Além disso, quando as células endoteliais são confluentes na superfície dos módulos há uma formação de tight junctions que pode ser visto por imunofluorescência da VE-caderina (Figura 2). Análise de transferência de massa demonstrou que os módulos são capazes de suportar altas densidades celulares (8x10 7 células / cm 3), sem desenvolver um núcleo necrótico devido ao transporte de oxigênio inadequada, o que muitas vezes é problemático em tecidos maiores (por exemplo, módulos de grande diâmetro, D = 1,4 mm) (Figura 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Figura 1" /> Figura 1: Módulo de fabricação e contração. Contração módulo ocorreu durante os três dias seguintes HUVEC-C (linhagem de células endoteliais) semeadura resultados diâmetro módulo significativamente menor e comprimento (p &lt;0,001) (A). Incorporados células HepG2 foram distribuídos uniformemente dentro dos módulos no momento da fabricação e manteve alta viabilidade (B). [Live células verdes; células mortas red] [adaptado de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Figura 2" /> Figura 2: VE-caderina coloração de imunofluorescência de junções celulares CE apertado 10 dias após RAEC onde revestidas na superfície de um módulo. Barra de escala = 50 mm. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Figura 3" /> Figura 3: coloração tricrômico de Masson de módulos típicos (0,76 mm de diâmetro inicial) e grandes módulos (1,4 mm de diâmetro inicial) demonstrar o efeito das limitações de difusão de oxigênio. Sete dias após a fabricação, um grande número de células mortas tinham formado dentro do núcleo das grandes módulos (painel inferior direito), deixando apenas uma fina borda (~ 200μm de espessura) de células viáveis. Por outro lado, os módulos pequenos retida uma distribuição uniforme e elevado de células vivas. [Módulos incorporado com células HepG2 e revestidos com células endoteliais.] [Extraído de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Figura 4" /> Figura 4: (A) HMEC-1 semeado em poloxamine - módulos de colágeno. Após o dia 1 de semear os módulos poloxamine colágeno mantêm a sua forma cilíndrica e não há propriedades apego limitado de células do colágeno em comparação com os módulos apenas. Barra de escala = 200 mM. (B) imagem do microscópio de luz de um módulo de colágeno contendo PLGA baseado em microesferas biodegradáveis. Barra de escala = 500 mm. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Figura 5" /> Figura 5: Exemplos de ensaios in vitro. (A) A angiogênese ensaio: capilar, como formações no módulo semeadas com células endoteliais podem ser facilmente detectados e quantificados após 5 dias de incubação com células-tronco derivadas de tecido adiposo. (B) imagens de microscopia confocal de um ensaio ao vivo / morto em módulos, onde o sFORMAÇÃO PARA células vivas é verde e de células mortas é vermelho. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Figura 6" /> Figura 6: Módulos de colágeno contendo embutidos células-tronco mesenquimais e uma camada superficial de células endoteliais. Módulos foram expostos a tensão de cisalhamento (~ 0,64 dyn / cm 2) por 7 dias em uma câmara microfluídicos e começar a mostrar a fusão em pontos de contato. Células endoteliais estão manchadas com vWF (marrom) e núcleos de células mesenquimais aparecem em azul (H &amp; E). Barra de escala = 100μm. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Figura 7" /> Figura 7: Centenas de módulos de colágeno contendo células-tronco derivadas de tecido adiposo (ASC) e revestidos com células endoteliais microvasculares Humanos (HMEC) são implantados sob a pele do rato para estudar ASC / HMEC interação in vivo para aplicações de regeneração de gordura.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

Não há conflitos de interesse declarados.

Nós fabricamos diversas micro-tecidos usando módulos integrados com diferentes tipos de células 1-11. Conseguimos incorporados cardiomiócitos primária, ilhotas, células-tronco adiposas e células mesenquimais do estroma, bem como várias linhagens de células incluindo HepG2, NIH 3T3 e clone 9 células do fígado. Temos os módulos revestidos com vários tipos de células endoteliais de aorta de ratos, incluindo células endoteliais, células endoteliais da veia umbilical humana e humanos células endot...

<table><tbody><tr><td>Intramedic tubing PE60</td><td>BD Biosciences</td><td>427416</td><td>Different diameter of tubing can be used to change the diameter of the modules</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>GIBCO, by Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>GIBCO, by Life Technologies</td><td>25200-072</td><td></td></tr><tr><td>Purcol acidificed collagen, 3 mg/mL</td><td>Cedarlane Labs</td><td>5005-B</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>20G needle</td><td>BD Biosciences</td><td>305175</td><td>Diameter of the needle needs to be similar to the diameter of the tubing</td></tr><tr><td>3 mL syringe</td><td>BD Biosciences</td><td>309585</td><td></td></tr><tr><td>15 mL tube</td><td>BD Biosciences</td><td>352096</td><td></td></tr><tr><td>50 mL tube</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>Convertors Self-Seal Pouch 7 1/2&amp;#8221; x 13&amp;#8221;</td><td>Cardinal Health</td><td>92713</td><td></td></tr><tr><td>10 ml Wide Tip serological pipet</td><td>BD Biosciences</td><td>357504</td><td></td></tr><tr><td>10 ml serological pipet</td><td>BD Biosciences</td><td>357551</td><td></td></tr><tr><td>5 ml serological pipet</td><td>BD Biosciences</td><td>357543</td><td></td></tr></tbody></table>

fabrication of micro-tissues using modules of collagen gel containing cells

Este protocolo descreve a fabricação de um tipo de micro-tecidos chamados de módulos. A abordagem módulo gera uniforme, tecidos escalável e vascularizada. Os módulos podem ser feitas de colágeno, bem como outros materiais gelable ou reticulável. Eles são cerca de 2 mm de comprimento e 0,7 mm de diâmetro sobre a fabricação, mas encolher de tamanho com células incorporado ou quando os módulos são revestidos com células endoteliais. Os módulos individualmente são pequenos o suficiente que as células incorporados estão dentro do limite de difusão de oxigênio e outros nutrientes, mas módulos pode ser embalado para formar tecidos maiores que são perfusable. Estes tecidos são modulares na construção, porque tipos diferentes de células pode ser incorporado em ou revestidos nos módulos antes de serem embalados para formar tecidos complexos. Há três passos principais para fazer os módulos: (1) neutralizar o colágeno e incorporação de células-lo, (2) gelificação do colágeno no tubo e corte os módulos e (3) revestimento dos módulos com células endoteliais.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Criação de micro-tecidos utilizando gel de colágeno cilíndricos, chamados módulos, que contêm células embarcados e cuja superfície é revestida com as células endoteliais.

Fabricação de Micro-tecidos utilizando Módulos de Colágeno Gel células contendo

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.7K visualizações.  University of Toronto. Criação de micro-tecidos utilizando gel de colágeno cilíndricos, chamados módulos, que contêm células embarcados e cuja superfície é revestida com as células endoteliais.

Vídeo: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

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

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Engenharia baseada em fibrina construções de tecido de miofibroblastos e de aplicação de restrições e tensão para induzir a organização das células e colágeno (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Um método aperfeiçoado para a preparação de colágeno tipo I Da Pele

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Fabricação de Miogênica Constrói engenharia de tecidos

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Biologia

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Camada por camada de colágeno Deposição em microfluídicos para Microtissue Estabilização

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

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 Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D para terapia regenerativa injetável e triagem de drogas de alto rendimento

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Microengenharia 3D Colágeno Hidrogéis com Alinhamento de Fibras de Longo Alcance

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...