Os autores gostariam de agradecer NSERC Discovery Grant 402462 &quot;andaimes da engenharia de tecidos para controlar pluripotentes induzidas comportamento de células-tronco&quot;.

Notas antes de iniciar o protocolo: O fibrinogênio é uma proteína derivada de sangue e treinamento de segurança, pois, adequado deve ser concluída antes do manuseio. Este protocolo requer 2 dias para ser concluído assim que o tempo das culturas-tronco desejadas de forma adequada para garantir que eles estão prontos para a semeadura. Em termos de cálculo quanto fibrinogénio a pesar, três pratos 35 mm de petri de solução salina tamponada com tris (TBS, pH 7,4) contendo 110-130 mg de fibrinogénio dissolvido em 3 mL de TBS será suficiente para produzir uma placa de 24 poços contendo 400 uL andaimes de fibrina em cada poço. 1. Dia: Fazendo soluções de fibrinogênio e diálise durante a noite <ol><li> Tome fibrinogênio liofilizado da geladeira e deixe descansar por 20 minutos para permitir que a temperatura ambiente. </li><li> Adicionar 3 ml de solução salina tamponada com Tris (TBS) a cada placa de Petri de 35 mm. Cada placa de rendimentos de fibrinogénio 2-3 mL de solução de fibrinogénio variando em concentração de 12-20mg / mL e da quantidade total de fibrinogénio necessária pode ser calculada com base na estas aproximações. </li><li> Pesar cerca de 100-130 mg de fibrinogénio (Fg) e salpique para a superfície do presente TBS em cada prato. Espere 5 minutos para que o fibrinogénio para começar a entrar em solução. Cubra placa de Petri com tampa. </li><li> Pratos de Petri incubar a 37 ° C durante 2 horas para permitir que o fibrinogénio em totalmente ir para dentro da solução de TBS. </li><li> Tubo de diálise Wet (7000 MW cortado) com TBS. Extremidade inferior dobra da extremidade da tubagem e se fecham com uma braçadeira de diálise. </li><li> Pipetar as soluções de fibrinogénio a partir de pratos em tubo de diálise. Dobre extremidade superior e se fecham com uma braçadeira de diálise. </li><li> Tubo de diálise contendo o local de solução de fibrinogénio em um recipiente de 4 litros de TBS. Solução mistura usando placa de agitação definido para a baixa velocidade (100 rpm). </li><li> Dialisar solução durante a noite em agitar placa durante, pelo menos, 12 horas. A solução de TBS não precisa de ser alterada ao longo deste período de tempo. </li></ol&gt; 2. Dia 2: Polimerização de andaimes de fibrina e semeadura de células estaminais em andaimes Todos os passos descritos para este processo deve ser realizado em um capuz estéril de cultura de tecidos como estes suportes serão semeada com culturas de células estaminais. <ol><li> Remover a solução de fibrinogénio a partir de tubo de diálise e coloque em um tubo de tamanho apropriado cónica (quer mL 15 ou 50 mL). </li><li> Solução de fibrinogénio filtro com um filtro de seringa de 5,0 mm a remover as impurezas grandes na solução. Dependendo do volume da solução, a utilização de filtros múltiplos pode ser necessário para processar a solução inteira. </li><li> Filtro estéril, a solução de fibrinogénio com um filtro de 0,22 um para um tubo de 50 mL cónico. Usando uma pipeta de tamanho adequado, determinar o volume da solução de fibrinogénio filtrou-se para uso posterior ao fazer cálculos. Dependendo do volume da solução, a utilização de filtros múltiplos pode ser necessário para processar a entsolução ira. </li><li> Medir a absorvância da solução de fibrinogénio a 280 nm utilizando o espectrofotómetro. Um factor de diluição de 50 vezes é necessário para obter uma absorvância inferior a 1. </li><li> Calcular a concentração de proteína presente na solução de fibrinogénio através da seguinte equação: [Fibrinogénio em mg / mL] = (A 280 × factor de diluição) ÷ 1,55 1,55 onde é o coeficiente de extinção a 280 A para o fibrinogénio humano 13. Em seguida calcular a quantidade de TBS necessário para diluir a concentração da solução a 11,1 mg / mL de fibrinogénio com base no volume inicial. A concentração final de proteína na andaimes de fibrina será de 10 mg / mL, após a polimerização. </li><li> Obter trombina estéril e soluções de cloreto de cálcio. Em primeiro lugar, adicionar 15 uL solução de trombina (concentração: 40 U / mL - concentração final no andaime será de 2 U / mL) para o lado direito de cada poço na primeira linha da placa de 24 poços. Em seguida, adicionar 15 uL de 50 mM de cálcio chlsolução oride para o lado esquerdo de cada poço. Finalmente, adicionar 270 uL da solução de fibrinogénio ao prato. Agitar placa de lado a lado, seguido por agitação trás para a frente para assegurar a mistura de soluções. </li><li> Deixe a linha que contém as misturas polimerizar durante 5 minutos à temperatura ambiente. Os andaimes tornam-se mais opaca como polimerização. </li><li> Repetir os passos 2.6 e 2.7 até que o número desejado de andaimes de fibrina foram polimerizados. </li><li> Após a incubação último minuto 5, colocar as placas dentro de uma incubadora a 37 ° C para permitir a polimerização completa de andaimes. </li><li> Após a incubação de uma hora é completa, o próximo passo é a semente da andaime com corpos embrióides. Usando um 20 pipettemen microlitro, selecione um corpo embrióides individual e lugar no centro do andaime de fibrina. Verifique com microscópio que cada poço contenha um corpo único embrióides. </li><li> Adicionar 5 uL de solução de trombina (concentração: 40 U / mL) e 5 uL de 50 mM de cloreto de cálciosolução no topo de cada corpo embrióides seguido por 90 uL de solução de fibrinogénio a cada poço. A solução deve formar uma bolha encapsulando os corpos embrióides. </li><li> Colocar a placa de volta em 37 ° C incubadora durante uma hora adicional para assegurar a polimerização. </li><li> Após a incubação, adicionar 1 mL de meio de cultura de células apropriadas para cada placa bem e de volta para o local de 37 ° C incubadora. </li></ol> 3. Os resultados representativos A Figura 1 mostra um diagrama esquemático do vista lateral de um indivíduo bem contendo o 3D fibrina sistema de cultura de andaime semeado com um corpo embrióides. Figura 2A mostra imagens representativas de rato células pluripotentes induzidas estaminais a ser cultivado em camadas alimentadoras de fibroblastos de rato embrionários. Estas células são então induzida a formar corpos embrióides, agregados de células que contenham progenitores neurais, utilizando um ácido retinóico 8 dias de tratamento protocolo (Figura 2B) como previously descrito 14 enquanto que a Figura 3 mostra o aspecto de um IPS-derivada corpo embrióides após 3 dias de cultura dentro de andaimes 3D de fibrina. Resultados semelhantes têm sido obtidos anteriormente usando células de ratinho estaminais embrionárias 7. Além disso, este método tem sido utilizado para a cultura IPS-derivados corpos embrióides produzido usando um protocolo diferente envolvendo ácido retinóico e purmorphamine 15 dentro de andaimes 3D de fibrina, demonstrando a versatilidade da presente sistema de cultura. <img alt="Figura 1." src="/files/ftp_upload/3641/3641fig1.jpg" /> Figura 1. Esquemático, mostrando a vista lateral de um indivíduo bem contendo um 3D fibrina andaime semeado com um corpo embrióides. <img alt="Figura 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> Figura 2. Rato tronco pluripotentes induzidas cultura de células (iPS) e diferenciação. Para diferenciar essas células em neurfenótipos al, as células iPS são cultivadas em suspensão para produzir agregados de células chamados corpos embrióides (EBS). Estes EBS são então tratadas com ácido retinóico para induzir a diferenciação neuronal e este protocolo foi previamente utilizado para induzir a diferenciação neuronal das células estaminais embrionárias. A) Indiferenciado rato colônia de células iPS cultivadas sobre uma camada de fibroblastos embrionários alimentador do mouse. B) corpos embrióides derivados de células de rato iPS em cultura em suspensão tomada no dia 8 após tratamento com ácido retinóico para induzir a diferenciação neuronal. Barra de escala é 100 mm. <img alt="Figura 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> Resultados Figura 3. Exemplo de um mouse iPS embrióides corpo após 3 dias de cultura dentro de um andaime 3D fibrina. As células começaram a migrar e se diferenciar dentro do andaime de fibrina. Barra de escala é 500 mm.

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Os autores não têm nada a revelar.

Este protocolo detalhado acima fornece um método para a geração de andaimes 3D de fibrina para a cultura de células estaminais pluripotentes, especificamente para o rato embrionário e células estaminais pluripotentes induzidas. Este sistema de cultura de 3D ​​biomaterial baseado com mais precisão imita o nicho de células-tronco encontrada in vivo e, como resultado, pode ser utilizado para rastrear sinais biológicos para determinar os seus efeitos sobre a diferenciação de células-tronco ...

Equipamento necessário: Balança analítica medidor de pH De cultura de tecidos incubadora (37 ° C, 5% de CO 2) Mexa placa Espectrofotômetro Capa cultura estéril tecido Solução salina tamponada com Tris (pH 7,4) (4 Necessita L mais suficiente para dissolver o fibrinogénio) 50 mM de solução de cloreto de cálcio Estéreis tubos cónicos (15 ou 50 mL) 35 mm placas de Petri Tubo de diálise (7.000 corte MW) Clipes de diálise 5.0 uM filtros de seringa Embalados individualmente estéril de 0,22 mM de seringas filtros Seringa 24 placas de cultura de tecidos <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Fibrinogénio (humano) </td><td> Calbiocbainha </td><td> 341578 </td><td></td></tr><tr><td> A trombina (humano) </td><td> Sigma </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

As células estaminais são encontrados em que ocorre naturalmente microambientes 3D in vivo, os quais são muitas vezes referida como a célula nicho haste 1. A cultura de células-tronco dentro de 3D ​​biomaterial andaimes fornece uma maneira para exatidão imitar estes microambientes, proporcionando uma vantagem sobre os métodos tradicionais de cultura 2D usando poliestireno, bem como um método para tecidos de engenharia de substituição 2. Enquanto polystrene cultura 2D tecido tem sido utilizada para a maioria das experiências de cultura de células, andaimes 3D biomaterial pode mais estreitamente replicar os microambientes encontrados in vivo, permitindo estabelecimento mais precisa da polaridade de células no meio ambiente e possuindo propriedades bioquímicas e mecânicas semelhantes a tecido mole. 3 A variedade de origem natural e andaimes biomaterial sintético têm sido investigados como ambientes 3D para apoiar o crescimento de células-tronco. Enquanto andaimes sintéticos podem ser sintetizados a ter uma maior range de propriedades mecânicas e químicas e muitas vezes têm uma maior reprodutibilidade, biomateriais naturais são frequentemente compostas de proteínas e polissacáridos encontrados na matriz extracelular e, como resultado contêm sítios de ligação para a adesão celular e prontamente suporte de cultura de células. Fibrina andaimes, produzido por polimerização do fibrinogénio proteína obtida a partir de plasma, têm sido amplamente investigados para uma variedade de aplicações de engenharia de tecidos tanto in vitro como in vivo 4. Tais suportes pode ser modificada usando uma variedade de métodos para incorporar os sistemas de libertação controlada para a entrega de factores terapêuticos 5. O trabalho anterior mostrou que andaimes tais pode ser utilizado com sucesso para a cultura de células estaminais embrionárias e este sistema de cultura de andaime baseado pode ser usado para examinar os efeitos de vários factores de crescimento sobre a diferenciação das células-tronco semeadas dentro 6,7. Este detalhes protocolo do processo de polymerizing de fibrina scaffolds a partir de soluções de fibrinogénio, usando a actividade enzimática da trombina. O processo leva 2 dias para se completar, incluindo um passo de diálise durante a noite para a solução de fibrinogénio para remover citratos que inibem a polimerização. Estes métodos detalhados contar com as concentrações de fibrinogênio determinados a ser o ideal para embrionário e induzida por cultura de células pluripotentes estaminais. Outros grupos têm investigado andaimes de fibrina para uma vasta gama de tipos de células e aplicações - demonstrando a versatilidade desta abordagem 8-12.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Isso detalhes o trabalho de preparação de fibrina scaffolds 3D para cultura e diferenciação das células-tronco plutipotent. Tais suportes podem ser usados ​​para examinar os efeitos de vários compostos biológicos sobre o comportamento das células estaminais, bem como modificada para conter sistemas de entrega de drogas.

Preparação de fibrina Andaimes 3D para aplicações de cultura de células-tronco

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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Bioengineering

25.1K visualizações.  University of Victoria. Isso detalhes o trabalho de preparação de fibrina scaffolds 3D para cultura e diferenciação das células-tronco plutipotent. Tais suportes podem ser usados ​​para examinar os efeitos de vários compostos biológicos sobre o comportamento das células estaminais, bem como modificada para conter sistemas de entrega de drogas.

Vídeo: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Bioengenharia

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Amostra Estratégias Preparação para Mass Spectrometry imagem da cultura celular 3D Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hidrogel Andaimes para articular de condrócitos Cultura e Geração de cartilagem

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Repovoamento celular epitelial e Preparação de Rodent Matriz Extracelular Andaimes para o desenvolvimento do tecido renal

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabricação de Mecanicamente Tunable e Bioativos metal andaimes para aplicações biomédicas

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

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 ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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 ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Síntese de biocompatíveis cristal líquido de elastómero Espumas como andaimes celulares para cultura de células 3D espaciais

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...