Este estudo foi realizado no programa dos materiais biomédicos (BMM), instituto de pesquisa. BMM é cofinanciado pelo Ministério Holandês de Assuntos Econômicos, Agricultura e Inovação. A contribuição financeira da Hartstichting Nederlandse é reconhecido agradecimento.

1. Cultura de células humanas veia safena <ol><li> Isolar células da veia safena magna, adquirido a partir de um doador de acordo com as orientações para utilização de material secundário, de acordo com o protocolo de Schnell et ai. 9 e armazená-las em azoto líquido. A partir da parte da veia safena magna peças cortadas a partir de um doador de 2 x 2 mm a cultura numa placa de seis poços. Use dois pedaços por poço. Geralmente, as células podem ser obtidas suficientes para encher frascos com cerca de 3 0,25 x 10 6 células em azoto líquido. HVSCs são caracterizados como miofibroblastos, mostrando a expressão de vimentina, sem expressão da desmina e uma subpopulação expressando α actina de músculo liso 10. Ainda iniciar o protocolo para descongelar as células a partir do azoto líquido para aumentar o número de células. </li><li> Colocar as células a partir de um frasco para dentro de um frasco de cultura de tecido T75 e adicionar meio de crescimento (GM), consistindo de DMEM avançada, 50 ml de soro fetal bovino (FBS), 5 mL de penicilina /estreptomicina e 5 ml de L-Glutamax. Mudar meio cada 2-3 dias. </li><li> Células crescem confluente aproximadamente após 14 dias. Loja 0,5 x 10 6 células num frasco em azoto líquido, referido como uma passagem. </li></ol> Nota: Congelamento das células não é necessária quando a colheita HVSCs, mas é usado apenas para armazenamento. <ol start="4"><li> Coloque os HVSCs de um frasco em frasco de cultura de um T175 tecido e adicione GM. Mudar meio cada 2-3 dias. Células crescem confluentes após cerca de 7 dias. Loja 3 x 10 6 células em um frasco em nitrogênio líquido, conhecido como passagem 2. </li><li> Coloque as células de um frasco em uma rollerbottle e adicione GM. Mudar meio cada 2-3 dias. Células crescem confluente aproximadamente após 8 dias. Uma rollerbottle contém cerca de 20-30 x 10 6 células. Células de cultura até a passagem 6. Não usar células de uma passagem superior a passagem 9, uma vez que o fenótipo celular pode mudar. </li></ol&gt; 2. Engenharia de construções de tecido à base de Fibrina <ol><li> Prepare a cola de silicone por mistura do elastómero com o agente de cura (10:1). Corte 7 x 3 segmentos retangulares mm de velcro. Cole o velcro em uma placa de cultura de 6 bem, com membranas flexíveis para formar uma cruz, e deixar um espaço quadrado de 3 mm entre as tiras de velcro. </li></ol> Notas: Só use o lado macio do velcro e enfrentar este lado para cima. Ao colar o velcro, apenas cobrir o velcro com cola de silicone, não espalhe cola por todo o bem. Uma vez que as placas de cultivo têm fundo da membrana de silicone, usar algo debaixo da placa bem para reforçar as membranas flexíveis, para assegurar a facilidade de colagem para a placa. <ol start="2"><li> Seca-se a cola de silicone durante a noite num forno a 60 ° C para assegurar o endurecimento da cola. Esterilizar por adição de EtOH a 70% aos poços e incubar durante 30 min. Lavar 3x com PBS e remover todo o PBS do wells eo velcro. Colocar sob UV, durante 30 minutos e mantenha estéril até à sua utilização. </li><li> Prepara meio de Engenharia de Tecidos (TM), que consiste em GM e 130 mg de ácido L-ascórbico 2-fosfato. </li><li> Adicionar 1 mg / mL de ácido amino-capróico ε (ACA) para o TM. ACA é utilizado para os primeiros 7 dias de cultura para evitar a degradação de fibrina a partir de 11. Alternativamente, a aprotinina pode ser usado. </li><li> Para evitar a formação de touceira, permitir fibrinogênio atingir a temperatura ambiente antes de abrir. Sem aglomerados fibrinogénio irá dissolver mais facilmente. Dissolve-se o fibrinogénio com uma concentração de 10 mg real de proteína / ml TM 7 suplementado com ACA. Filtro estéril a solução de fibrinogênio, vários filtros podem ser necessários. Guarde em gelo até o uso. </li></ol> Nota: Para dissolver mistura de fibrinogénio com cuidado para evitar a formação de espuma muito. <ol start="6"><li> Dissolve-se trombina para uma concentração de 10 Ul / ml TM 7 suplementado com ACA. Guarde em gelo até o uso. </li> &lt;/ Ol&gt; Nota: Armazenamento em gelo é necessário para evitar a gelificação precoce de trombina eo fibrinogênio. <ol start="7"><li> Trypsinize as células, ressuspender em GM e contagem. </li><li> Usar 15 x 10 6 células / ml para semear as construções de tecido à base de fibrina (concentração com base em métodos de engenharia de tecidos de válvulas cardíacas 7). Um único gel consiste em 100 ul de gof el e, assim, de 1,5 x 10 6 células. Colocar 1,5 x 10 6 células num tubo de centrífuga, resultando em tantos tubos de centrifugação como o número de geles que serão feitas. </li><li> Centrifugar as células a 350 xg durante 7 minutos e descarta-se o sobrenadante. Ressuspender as células em 50 ul de trombina. Adicionar 4 ml de azul fluorescente microesferas de poliestireno a esta suspensão. Colocar 50 mL de fibrinogénio num frasco. Usar uma pipeta para assumir a trombina 50 uL de células. Aumento do volume da pipeta de 100 uL. Pipete 50 pi da solução de trombina comcélulas no frasco com o fibrinogênio para misturar a trombina eo fibrinogênio, e levar até a mistura de 100 ml. </li></ol> Nota: A fluorescente microesferas de poliestireno são utilizados como marcadores de referência internos para análise de imagem. Quando a mistura de trombina e fibrinogénio, evitar a formação de bolhas de ar, pipetando cuidadosamente a mistura. As bolhas de ar resultará em furos no gel de fibrina. <ol start="10"><li> Pipeta a mistura de gel dentro e entre as tiras de velcro. O tempo típico para a gelificação do gel de fibrina, uma vez que os componentes são misturados, é da ordem de 20 segundos. </li></ol> Notas: Para fazer isso, o mais rapidamente possível para evitar a gelificação antes da mistura é pipetados na placa bem. Pratique antes de usar células e miçangas. <ol start="11"><li> Incubar as suspensões durante 30 minutos a 37 ° C numa atmosfera humidificada de 95% / 5% de CO 2 incubadora para permitir a gelificação antes de adicionar a cultura TM com ACA. </li><li> Substitua TM com ACA cada 2-3 dias para os primeiros 7 dias. Géis são estáveis ​​o suficiente depois de uma semana a ser cultivadas sem ACA. Depois da primeira semana TM substitua cada 2-3 dias. Adicionar 6 ml de TM por poço. No dia 12 células produziram colágeno suficiente para visualização. </li></ol> 3. Aplicando tensão e induzir mudanças no Strain e Restrições <ol><li> Estirpe estático é aplicado directamente pelas células, devido à tracção e célula de compactação 12. Para induzir a reorganização do colágeno, cortar o tecido construir solta de duas tiras de velcro em uma direção. Isto resulta em limitações unidireccionais (Figura 1A). Executar esta no dia 12, uma vez que a construção é então suficientemente estável e colagénio tenha sido depositado. </li><li> Aplicar a tensão cíclica mediante a aplicação de vácuo ao fundo das placas de cultura com membranas flexíveis, com a utilização do sistema FX4000T Flexcell. Colocar as placas em uma placa de base, em cima dos postos de carregamento (que são uma parte do diae dispositivo de carga cíclica). A aplica-se uma bomba de vácuo sobre a membrana e, assim puxa a membrana sobre o posto rectangulares encontra-se por baixo. Devido aos elementos de montagem em forma de cruz no tecido constrói à membrana (por intermédio do velcro) e o posto rectangulares da estirpe cíclico aplicado é uniaxial (Figura 1B). </li><li> Inicialmente, usar cultura estática, durante 5 dias para atingir a integridade mecânica inicial, antes da aplicação da estirpe cíclico, começando no dia 6. Programa de um protocolo de mancha cíclico para o controlador para a bomba de vácuo. Por exemplo, usar um protocolo estirpe cíclico intermitente previamente estabelecida, com o sentido 13 uniaxial. Este é constituído por uma estirpe intermitente de uma onda sinusoidal com uma Hz, esticando de 0 a 5% de deformação, por períodos de 3 horas, alternadas com períodos de descanso 3 hr. Realizar esse protocolo tensão cíclica por 7 dias, induzindo uma organização de colágeno alinhados. </li><li> Tipicamente, após 12 dias HVSCs ter produzido uma organizatio colagénio alinhadosn. Depois de uma organização do colagénio alinhado é atingido, mudar a direcção de tensão uniaxial cíclico, para ser perpendicular à direcção da tensão inicial. Para fazer isso, transformando os cargos retangulares em 90 °. </li></ol> 4. Visualizando Células e colágeno <ol><li> Para visualizar ativa, remodelação do colágeno em tempo real, as amostras de etiquetas com sondas que não interferem com a viabilidade das células ou a formação de colágeno. Use sondas de mancha fluorescente do citoplasma da célula e colágeno. </li><li> Alternativamente segunda geração harmónica utilizando a microscopia confocal a laser pode ser usada para visualizar as estruturas de colagénio utilizando autofluorescência 14, com excitação a 780 nm e entre 500-550 nm de detecção. </li><li> Retire as placas de cultura a partir da configuração e da incubadora, para as amostras ciclicamente tensas durante o período de descanso de 3 horas, e transportá-los para um microscópio de varredura a laser confocal para visualizar as células e colágeno. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

O sistema modelo descrito de construções de fibrina por células povoadas tem um grande potencial para o estudo de células e colagénio (re) organização (De Jonge et ai. 15), por exemplo, para ser utilizado para fins de engenharia de tecidos. Ao usar fibrina, como o transportador inicial da célula, depois da degradação da fibrina, um tecido é criado com apenas células e matriz endógena. Desta forma, as células são estimuladas para reagir à tensão, estático ou de natureza cíc...

Tecidos cardiovasculares têm uma função de suporte de carga de destaque. Em particular, o conteúdo ea organização das fibras de colágeno na matriz extracelular contribuem para as propriedades de suporte de carga e dominar a força do tecido total 1. Na engenharia de tecidos condicionado mecânica da construção é usada - geralmente constituídos por (cíclica) regimes esforçando - para melhorar a organização do tecido e propriedades mecânicas 2,3. Entendimento completo da organização do colagénio induzida por deformação em geometrias complexas para criar tecidos tecidos de colagénio com uma arquitectura predefinida ainda não foi alcançada. Isto é principalmente devido ao nosso conhecimento limitado de remodelação do colágeno nos tecidos em desenvolvimento. Modelos existentes, principalmente, dar informações sobre o resultado líquido final de remodelação do colágeno com o uso de pressão estática 4-6. Aqui nós fornecemos um sistema modelo altamente ajustável que permite o estudo da organização do colagénio (re) de uma forma em tempo real, em 3D, sob influênciada estirpe estático ou cíclico. As construções de tecido são à base de fibrina, assegurando que toda colagénio no construto é endógena. Organização celular e colágeno nas construções é visualizado, e um sistema de referência interna nos permite mudar as células e estruturas de colágeno para a análise de time-lapse. Neste protocolo, iremos descrever o uso do sistema de modelo de veia safena Humanos Células (HVSCs), uma vez que estas células são conhecidos pela sua produção aumentada de matriz extracelular e capacidade para remodelar a matriz e a utilização conhecida em engenharia de tecidos cardiovasculares 7, baseado na obra de Jonge et al. oito

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

Conteúdo de colágeno e organização no desenvolvimento de tecidos de colágeno pode ser influenciada por cepas do tecido local e restrição dos tecidos. Engenheiros de tecidos pretendem usar esses princípios para criar tecidos com arquiteturas de colágeno pré-definidos. A plena compreensão dos processos subjacentes exatas de remodelação do colágeno para controlar a arquitetura do tecido final, no entanto, é inexistente. Em particular, pouco se sabe sobre a orientação a (re) das fibras de colagénio, em resposta a alterações nas condições de carga mecânica dos tecidos. Nós desenvolvemos um sistema em modelo in vitro, que consiste em biaxialmente limitados miofibroblastos semeado construções de fibrina, para elucidar ainda mais colágeno (re) orientação em resposta ao i) reversão biaxial para condições de carga estática uniaxiais e ii) carga cíclica uniaxial do biaxialmente constrangido construções, antes e depois de uma mudança de direcção de carga, com a utilização do dispositivo de carregamento FX4000T Flexcell. Time-lapse confocal de imagem é utilizado para visualize colagénio (re) orientação de uma maneira não destrutiva. Organização celular e colágeno nas construções podem ser visualizados em tempo real, e um sistema de referência interna nos permite mudar as células e estruturas de colágeno para a análise de lapso de tempo. Vários aspectos do sistema do modelo podem ser ajustados, como fonte de células ou a utilização de células saudáveis ​​e doentes. Aditivos podem ser utilizados para elucidar os mecanismos subjacentes a remodelação do colagénio, por exemplo, por adição de MMPs ou bloqueio integrinas. A forma eo tamanho da construção pode ser facilmente adaptada para necessidades específicas, resultando em um sistema modelo altamente ajustável para o estudo de células e colagénio (re) organização.

Este modelo de sistema permite a cultura myofibroblast-semeados gel de fibrina. Figura 1A mostra um tecido cultivado pela primeira vez sob restrições biaxiais estáticas. Constrangimentos de tecido são libertadas por corte do gel de fibrina a partir de duas restrições, para criar restrições estáticas uniaxiais e compacta do tecido e remodela depois (Figura 1A). Para estirpe cíclico, o tecido é cultivada sob constrangimentos biaxiais estáticos também. Após 5 dias de tensão ...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

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

Este modelo de sistema começa a partir de um gel de fibrina miofibroblastos povoada que pode ser usado para estudar colagénio endógeno (re) organização em tempo real de uma forma não destrutiva. O modelo de sistema é muito sintonizável, como ele pode ser usado com diferentes fontes de células, aditivos médio, e pode ser facilmente adaptada às necessidades específicas.

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 ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K visualizações.  Eindhoven University of Technology.  Este modelo de sistema começa a partir de um gel de fibrina miofibroblastos povoada que pode ser usado para estudar colagénio endógeno (re) organização em tempo real de uma forma não destrutiva. O modelo de sistema é muito sintonizável, como ele pode ser usado com diferentes fontes de células, aditivos médio, e pode ser facilmente adaptada às necessidades específicas. 

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

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Dirigido Cellular Automontagem para Fabricar Anéis Cell-Derived Tecido para Análise Biomecânica e Engenharia de Tecidos

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Bioengenharia

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sem contato, Monitoramento Label livre de células e matriz extracelular utilizando Espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Os tecidos músculo esquelético de engenharia a partir de células progenitoras mioblastos murinos e Aplicação da estimulação elétrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Engenharia de Tecidos de tumor estromal Microenvironment com Aplicação a invasão Cancer Cell

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation 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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Tratamento de Estruturas Ligamentares com Soro Condicionado por Exercício: Um Modelo Translacional de Engenharia de Tecidos

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Recombinante colágeno peptídeo Microcarriers para expansão da célula e seu potencial uso como sistema de entrega de células em um bioreator modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Uma nova técnica de sutura tenorrhaphy com enxerto de colágeno projetado por tecido para reparar defeitos grandes do tendão

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioink extracelular derivado da matriz de tecido sipâncreas para a impressão de construções de tecidopanpático 3D Carregadas de células

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...