This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

1. Preparação da solução de colagénio nativo solúvel <ol><li> Prepare comprar ou 200 mg de acidificada, solúvel, de colagénio tipo I da cauda de ratos a 1-3 mg / 15 ml, utilizando protocolos de isolamento convencionais, tais como relatado por Piez et ai. </li><li> Escala a quantidade de material de partida com base no volume final desejado de soluções de colagénio modificados. Aproximadamente 25-30 ml de fazer metilado e 25-30 ml de soluções de colagénio succiniladas, cada um em 3 mg / ml, a partir de 200 mg de colagénio nativo solúvel. </li></ol> 2. O colágeno metilação <ol><li> Dilui-se 100 mg da solução nativa, acidificou-se (pH 2-3) de colagénio a uma concentração de 0,5 mg / ml com água estéril gelada e manter a solução em gelo para evitar a gelificação. </li><li> Ajuste o pH da solução de colagénio para 9-10 com algumas gotas de NaOH 1 N e agita-se à TA durante 30 min. Observe-se o precipitado de colagénio, fazendo com que a solução se tornar turva. </li&gt; <li> Girar a solução de colágeno precipitado a 3000 × g durante 25 min. Um precipitado claro, semelhante a gel deve ser visível no fundo dos tubos. Aspirar e descartar adequadamente o líquido sobrenadante. </li><li> Ressuspender o precipitado de colagénio em 200 ml de metanol com HCl 0,1 N e permitir que a reacção de metilação para ocorrer com agitação à temperatura ambiente durante 4 dias. O colágeno não vai dissolver, mas deve se quebrar em pedaços muito pequenos visíveis que irão tornar a solução turva. </li><li> Após a metilação, centrifugar a solução a 3000 × g durante 25 min para sedimentar o colagénio metilado. Aspirar e descarte do sobrenadante metanol acidificado. </li><li> Dissolve-se o colagénio metilado em 25 ml de PBS estéril, dando uma concentração de aproximadamente 3 mg / ml, com uma pipetagem repetida, e filtrar a solução através de um filtro de células de 60 mm. Ajuste o pH da solução para 7,3-7,4 usando 20 ul incrementos de 1 N NaOH. </li><li> Avaliar a Concentraçãn da solução utilizando um kit de ELISA comercial colágeno rato ou kit de ensaio de hidroxiprolina. Dilui-se a solução a 3 mg / ml com PBS estéril. </li><li> Esterilizar a solução de colagénio metilado, transferindo-a para um frasco de vidro com uma tampa de rosca, de camadas cuidadosamente 3 mL de clorofórmio, na parte inferior da garrafa, permitir que a garrafa para definir O / N a 4 ° C, e em seguida assepticamente remover e armazenar o camada superior, que é o colagénio metilado. </li><li> Guardar a solução a 4 ° C para utilização no prazo de 1 mês. </li></ol> 3. O colagénio succinilação <ol><li> Dilui-se a outros 100 mg da solução nativa, acidificou-se (pH 2-3) de colagénio a uma concentração de 0,5 mg / ml com água estéril gelada e manter a solução em gelo para evitar a gelificação. </li><li> Ajuste o pH da solução de colagénio para 9-10 com algumas gotas de NaOH 1 N e agita-se à TA durante 30 min. O colagénio deve precipitar, fazendo com que a solução se tornar turva. </li><li> Dissolve-se 40 mg de sucanidrido cínico (0,4 mg por mg de colagénio) em 10 ml de acetona (1/20 do volume da solução de colagénio). Lentamente (em cerca de 0,5 ml incrementos) adicionar esta mistura à solução de colagénio com agitação, enquanto se monitorizava continuamente o pH. Manter o pH acima de 9,0 através da adição de 1 ou 2 gotas de NaOH 1 N conforme o pH se aproxima de 9,0. </li><li> Continuar a agitar à temperatura ambiente durante 120 minutos após a adição de todo o anidrido succínico em acetona. Observe a solução para se tornar claro como o colágeno succinilado dissolve. Verifique periodicamente o pH para assegurar que ela permaneça acima de 9,0. </li><li> Ajustar o pH da solução para 4,0 com 20 ul incrementos de 1 N de HCl. Observar a solução turva novamente, como o colagénio precipita succinilada. </li><li> Após a succinylation, centrifugar a solução a 3000 × g durante 25 min para sedimentar o colagénio succinilado. Aspirar e descarte do sobrenadante acidificou-se com anidrido succínico não reagido. </li><li> Dissolve-se o colagénio succiniladoem 25 ml de PBS estéril, dando uma concentração de aproximadamente 3 mg / ml, com uma pipetagem repetida, e filtrar a solução através de um filtro de células de 60 mm. Ajuste o pH da solução para 7,3-7,4 usando 20 ul incrementos de 1 N NaOH. </li><li> Avaliar a concentração da solução, utilizando um kit de ELISA comercial de rato de colagénio ou kit de ensaio de hidroxiprolina. Dilui-se a solução a 3 mg / ml com PBS estéril. </li><li> Esterilizar a solução de colagénio succinilado, transferindo-a para um frasco de vidro com uma tampa de rosca, de camadas cuidadosamente 3 mL de clorofórmio, na parte inferior da garrafa, permitir que a garrafa para definir O / N a 4 ° C, e em seguida assepticamente remover e armazenar o camada superior, que é o colagénio succinilado. </li><li> Guardar a solução a 4 ° C para utilização no prazo de 1 mês. </li></ol> 4. Verificação do colagénio Modificações <ol><li> Preparar amostras de 1 ml a partir de cada uma das soluções de colagénio nativo, metilados, e succiniladas e diluir para um coneentration de 0,1 mg / ml com água pura esterilizada para titulação de íons de hidrogênio. Diluir o tampão de pelo menos 1.000 vezes por diálise contra água através de um corte de 10 kDa membraneusing 3 mudanças de solução de pelo menos 4 horas cada. </li><li> Ajustar o pH das soluções para 7.3 com pequenas quantidades de HCl e de NaOH. Usando pH 7,3 como uma referência arbitrária, executar titulação de iões de hidrogénio em cada uma das amostras, como descrito por Tanford 16 para criar curvas de titulação para as soluções de colagénio nativo, metilados, e succinilada. </li><li> Traça-se a mudança de pH por volume de ácido adicionado em relação ao número de iões H + ligada por molécula. O pH elevado &quot;amino&quot; intervalo deve mostrar uma mudança no colagénio succinilado em direcção ao ponto neutro (uma perda de grupos amina), e o pH baixo &quot;carboxilo&quot; intervalo deve mostrar um deslocamento para a esquerda no colagénio desnaturado (uma perda de grupos carboxilo ) e um desvio para a direita na colagénio succinilada (um ganho no grupo carboxilos), em comparação com o colagénio nativo. </li><li> Avaliar a eficácia da reacção succinylation pela determinação da% de grupos amino no colagénio nativa substituído por succinylation usando o método colorimétrico de ácido 2,4,6-trinitrobenzenossulfónico (TNBA), seguindo protocolos padrão 17,18. </li></ol> 5. fabricação de dispositivos microfluídicos e propagação de celular <ol><li> Fabricar dispositivos microfluídicos usando métodos padrão 9. Use réplica moldagem de PDMS de SU-8 mestres de silício definido usando fotolitografia para criar câmaras de cultura de células de microfluidos com 100 um de altura, 0,4-1,5 mm de largura e 1-10 mm de comprimento canais para o crescimento celular. </li><li> Use um limpador de plasma para oxidar as superfícies do dispositivo e uma lâmina de vidro, em seguida, pressione em conjunto para ligação. Após a esterilização do dispositivo em caso de exposição à luz UV durante pelo menos 30 min, encher a câmara com 50 ug / ml de fibronectina em PBS estéril e incubar a 37 ° C durante 45 min. </li&gt; <li> Semear o dispositivo com as células, tais como de rato primária ou hepatócitos humanos, que necessitem de um gel de colagénio para a estabilização do fenótipo ou do estado de diferenciação. Usamos 20 ul de recém-isolados hepatócitos primários de rato 7,19 comercialmente disponíveis ou criopreservados hepatócitos humanos primários semeadas a 14 x 10 6 células / ml para cada dispositivo. </li><li> Permitir que as células para anexar para 4-6 horas, e, em seguida, lavar a células não ligadas e substituir a mídia chapeamento com meios de crescimento. Incubar O / N para assegurar célula completa divulgação e criar uma monocamada confluente de células. </li></ol> 6. Camada por camada de colágeno Deposição <ol><li> Em um fluxo laminar capuz de cultura de tecidos, volumes suficientes de preparar soluções de colagénio desnaturado e succiniladas de 10 aplicações de solução por cada dispositivo, bem como de alguns ml de meio. Mantenha as soluções no gelo. Nós usamos 20 jul (10-15 vezes o volume total de dispositivo) de colágeno por camada por dispositivo. </li><li> Serdescaroçamento com a solução metilado (policatiónico), suplente rubor os dispositivos com 20 l de metilado e depois succinilado soluções de colágeno, esperando 1 min entre cada aplicação. Lave o dispositivo de um total de 10 vezes por solução, que deve levar cerca de 20 min. Trabalhar rapidamente para minimizar a quantidade de tempo que as células são sem meios. </li><li> Observar colagénio lentamente acumulam à entrada / saída em função do tamanho. Se a resistência ao fluxo do fluido aumenta, lave o dispositivo uma ou duas vezes com a mídia, e depois continuar estratificação. </li><li> Após a aplicação de todas as camadas, lavar duas vezes com o dispositivo de meios frescos e retornar para a incubadora. Dependendo do tipo de célula, as alterações morfológicas induzidas extracelulares da matriz, tais como a polarização aumentada em hepatócitos, deve ser visível dentro de algumas horas. </li><li> Prepare dispositivos representativos para microscopia electrónica de transmissão usando métodos padrão de 9 para verificar a presença de uma matriz de colagénioa montagem no topo das células cultivadas. </li></ol> 7. Estabilização do fenótipo celular e função <ol><li> Imagem da morfologia celular, viabilidade, e a polaridade usando métodos padrão para o tipo de célula. Para hepatócitos, coletar imagens ao longo de 14 dias, utilizando microscopia de fase, coloração Live / Dead para a viabilidade, e corante CMFDA para a polarização apical para demonstrar os efeitos da deposição de colágeno. </li><li> Para a morfologia das células, lave o dispositivo através da entrada, com uma pipeta, com 20 ul de PBS para enxaguar, injectar 20 ul de PBS, e a imagem do dispositivo usando microscopia de contraste de fase. </li><li> Para a coloração Live / Dead, irrigue o dispositivo através da entrada com uma pipeta com 20 l de PBS para lavar, injetar 20 ml de solução de coloração Live / Dead com DAPI preparado seguindo as instruções do fabricante, incubar por 30 min a 37 ° C, lavar a Dispositivo de novo com 20 ul de PBS, e a imagem do dispositivo usando a microscopia de fluorescência. </li><li> Para bile canalicoloração Culi, lave o dispositivo através da entrada, com uma pipeta, com 20 ul de PBS para enxaguar, injectar 20 ul de 2 gotas de solução corante uM CMFDA com DAPI preparados de acordo com as instruções do fabricante, incubar durante 30 minutos a 37 ° C, lavar novamente o dispositivo com 20 ul de PBS, e a imagem do dispositivo usando microscopia de fluorescência. </li><li> Recolher os meios gastos e medir produtos metabólicos celulares em pontos de tempo apropriados ao longo da duração da cultura para determinar a função das células. Para hepatócitos, medir a quantidade de albumina e ureia na mídia gastos. </li><li> Realizar análises funcionais de ponto final nas próprias células, como as que avaliam os níveis de enzimas de atividade, coloração de anticorpos, ou análise de RNA. Para hepatócitos, induzir e medir a atividade das enzimas do citocromo P450 ou fase II conjugação enzima glutationa S-transferase. </li></ol>

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The authors have nothing to disclose.

Ultrafinos assembleias de colágeno puro pode ser depositado em células carregadas ou superfícies de material usando deposição de camada por camada de colágenos modificados. Os resultados deste estudo demonstram que a metilação e succinylation de colagénio nativo criar soluções policatiónicos e polianiónicos de colagénio (Figura 1) que pode ser utilizado com a técnica de camada-a-camada para depositar conjuntos de matriz de colagénio ultrafinos em células (Figura 2) ou o...

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table><tbody><tr><td>collagen type I, rat tail</td><td>Life Technologies</td><td>A1048301</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>Sigma-Aldrich</td><td>C3867-1VL</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>EMD Millipore</td><td>08-115</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>R%D Systems</td><td>3440-100-01</td><td>option for concentrated rat tail collagen</td></tr><tr><td>succinic anhydride</td><td>Sigma-Aldrich</td><td>239690-50G</td><td>succinylation reagent</td></tr><tr><td>anhydrous methanol</td><td>Sigma-Aldrich</td><td>322415-100ML</td><td>methylation reagent</td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td>pH precipitation reagent</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>320331-500ML</td><td>pH precipitation reagent</td></tr><tr><td>rat collagen type I ELISA</td><td>Chondrex</td><td>6013</td><td>option for detecting collagen content</td></tr><tr><td>hydroxyproline assay kit</td><td>Sigma-Aldrich</td><td>MAK008-1KT</td><td>option for detecting collagen content</td></tr><tr><td>hydroxyproline assay kit</td><td>Quickzyme Biosciences</td><td>QZBtotcol1</td><td>option for detecting collagen content</td></tr></tbody></table>

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.

Colagénio nativo pode ser modificado usando metilação e succinylation para criar soluções de colagénio policatiónicos e polianiónicos para uso na deposição de camada por camada. Succinylation modifica os grupos e-amino de colagénio nativo com grupos succinilo, metilação e modifica os grupos carboxilo de colagénio nativo com um grupo metilo (Figura 1A). Estas modificações às cadeias laterais de aminoácidos da proteína de colagénio alterar as curvas de titulação para pH das soluçõe...

Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

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

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 ...

layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.3K visualizações.  Massachusetts General Hospital, Harvard Medical School. 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.

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

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

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

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 ...

Bioengenharia

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Plataformas microfabricated para cultura de células mecanicamente dinâmico

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

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 ...

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. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Técnica microfluídico genipina Deposição de Cultura Extensão da micropatterned vasculares musculares Filmes Finos

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e Montagem de microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de microfluidic Bioprinting para a engenharia de tecidos e Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Uma plataforma Microfluídico para estimular condrócitos com compressão dinâmica

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Desenvolvimento de tecido cardíaco humano organizado 3D dentro de uma plataforma microfluida

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...