Agradecemos a George Giudice e Steven Eliason por doarem fibroblastos dérmicos humanos e Ramesh Raghupathy por ajuda com o algoritmo de rastreamento de cepas. O apoio a este trabalho foi fornecido por um Departamento de Educação do Departamento de Educação dos EUA em Áreas de Bolsa de Necessidade Nacional (GAANN P200A120071).

1. Preparação de estêncil
Prepare um estêncil no Parafilm para layout da localização de cada explanta, seguindo a geometria desejada (Figuras 1A e 1B). Espaço cada explante aproximadamente 1-2 mm de distância. Essa distância corresponde a um espaçamento ideal para gerar alinhamento de fibras entre explantas. Fixar o estêncil sob a área da tampa onde a amostra será preparada com fita.
2. Esterilização
Limpe completamente todos os componentes do bioreator usando 70% de etanol e esterilize por 2-3 horas sob luz UV antes da experimentação. Se um vaso alternativo estiver sendo usado em vez de um bioreator, então devem ser utilizadas técnicas adequadas de esterilização. Veja o passo 4.12 para comentários sobre o uso de uma placa de Petri de fundo de vidro.
3. Preparação do gel de fibrina
Após a esterilização dos componentes do bioreator, coloque uma fina camada de gel de fibrina na superfície da almofada. Detalhes para a preparação de soluções de estoque de fibrinogen e trombina para a fabricação de géis fibrinas de 6,6 mg/ml são descritos em Sander et al. 13 Um protocolo semelhante por Ye et al. 19 para fazer 3,3 mg/ml de géis fibrinas também podem ser vistos no site da JoVE.
<ol><li>Prepare uma solução de microesferas fluorescentes em DMEM a uma concentração de 10 milhões de contas/ml. As contas serão usadas para ajudar a rastrear deslocamentos de gel. Para alcançar essa concentração, combine 0,017 ml de solução de estoque de microesferas e 0,149 ml de DMEM em um tubo de microcentrifuge.</li>
	<li>Sonicar esta suspensão por 10 minutos para dispersar as contas e homogeneizar a solução.</li>
	<li>Solução Fibrin — Em um tubo C de 15 ml, misture 0,22 ml de solução de estoque de fibrinogênio com 0,44 ml de tampão HEPES de 20 mM. Adicione os 0,1667 ml de DMEM com microesferas criadas na etapa 3.1.</li>
	<li>Solução Thrombin — Em um tubo c separado de 15 ml, misture 0,0328 ml de solução de estoque de trombina, 0,131 ml de tampão HEPES de 20 mM e 0,00246 ml de 2 M CaCl2.</li>
	<li>Misture cuidadosamente a solução de trombina (passo 3.4) com a solução fibrinogênio (etapa 3.3) por tubulação para cima e para baixo de 5-10x até que a solução seja distribuída uniformemente. Evite introduzir bolhas o máximo possível. Para reduzir a quantidade de bolhas produzidas, tenha cuidado para não descarregar totalmente a pipeta durante a mistura.</li>
	<li>A adição de trombina fará com que a solução geleia rapidamente (~30 seg). Pipete a solução mista na tampa o mais rápido possível. Deixe o gel polimerizar em RT.</li>
	<li>Sele o bioreator, insira os blocos de aquecimento e conecte os termopares ao controlador de temperatura. Incubar o gel a 37 °C por 15-30 min.</li>
</ol>4. Preparação de explanta celular
<ol><li>Remova o meio do frasco T-75 contendo as células do fibroblasto dérmico humano.</li>
	<li>Enxágue cuidadosamente a superfície com aproximadamente 5 ml de salina tamponada de fosfato (PBS) para remover proteínas séricos. Adicione 1 ml de trippsina-EDTA e incubar por 3 minutos, ou até que as células tenham levantado.</li>
	<li>Depois que as células tiverem sido levantadas, gire a suspensão para baixo em uma centrífuga a 200 x g por 5 min. Remova o supernasce e resuspense a pelota em um volume de DMEM que permitirá uma concentração final de 20 milhões de células/ml.</li>
	<li>Enquanto as células estão girando para baixo na centrífuga desconecte o bioreator dos blocos de aquecimento e dos termopares. Transfira o bioreator para um armário de biossegurança e remova cuidadosamente a tampa seguindo técnicas ascetônicas.</li>
	<li>Crie explantas ao pipetar 0,3 μl da suspensão celular no gel de fibrina polimerizado, seguindo o padrão no estêncil. Cada explanta deve conter aproximadamente 6.000 células. Certifique-se de que são usadas dicas de micropipette de baixo volume (0,1-10 μl).</li>
	<li>Permitir que as células se instalem e se conectem à matriz de fibrinas por 1 hora a 37 °C.</li>
	<li>Com o bioreator ainda aberto, adicione aproximadamente 5 ml de DMEM suplementado com soro bovino fetal de 10% (FBS), 1% penicilina-estreptomicina, 0,1% anfotericina B e 10 mg/ml de aprotinina diretamente na câmara bioreatorial. O DMEM é tamponado bicarbonato e requer 5% de CO2 para manter um pH neutro. Uma vez que o bioreator não é fornecido com CO2, condiciona o meio em uma incubadora com 5% de CO2 por 2-3 horas antes do uso. Aprotinina é um inibidor de protease serino que é amplamente utilizado para reduzir a taxa de degradação fibrina20.</li>
	<li>Resseque o bioreator. Use uma seringa para fornecer um adicional de 5 ml de meio condicionado de CO2 através do encaixe farpado na porta de entrada. Dispense o meio lentamente e certifique-se de que todo o volume da câmara bioreatorial esteja preenchido. Remova cuidadosamente bolhas que se formam no bioreator.</li>
	<li>Fornecer alimentos frescos, 5% de CO2 condicionado ao bioreator durante todo o experimento, a fim de manter o pH, fornecer nutrientes e remover resíduos. Configure uma bomba de seringa com uma seringa de 10 ml ou 30 ml preenchida com 5% de CO2 médio condicionado. Conecte a seringa diretamente à porta de entrada na tampa bioreaator com tubos estéreis equipados com luer. Remova o encaixe masculino e conecte a tubulação ao encaixe farpado no bioreator (ver Figura 1C).</li>
	<li>Defina a taxa de perfusão para 0,01 ml/min. Conecte pedaços modificados de tubulação em ambas as portas de tomada e coloque as extremidades em um béquer de 100 ml para coletar resíduos.</li>
	<li>Use uma tomada de laboratório para definir as alturas das rações de entrada e saída para que um diferencial de pressão não se desenvolva no bioreator (consulte a Figura 1D para referência).</li>
	<li>Se um bioreator não estiver disponível, prepare amostras em placas de Petri de fundo de vidro de 35 mm com topos de vidro. Use um com um tamanho de deslizamento de cobertura que seja otimizado para o conjunto específico de objetivos a serem utilizados. As amostras que são preparadas em placas de Petri devem ser mantidas em uma incubadora a 37 °C e 5% de CO2. Nota: O poliestireno despolariza a luz e interferirá com a imagem dic, por isso os topos de vidro devem ser usados se a imagem DIC for conduzida. O contraste de fase é uma modalidade de imagem alternativa adequada. A transferência de pratos entre a incubadora e o microscópio dificultará o registro de imagens. Se as imagens não estiverem devidamente registradas, a cepa calculada na Seção 6 não será precisa.</li>
</ol>5. Imagem de lapso de tempo
<ol><li>Uma vez que a amostra tenha sido preparada, resseque o bioreator, reconecte os blocos de aquecimento e os termopares, e coloque a temperatura do bioreator a 37 °C.</li>
	<li>Coloque o bioreator no estágio motorizado montado no microscópio. Posicione o objetivo DIC 20X sob a porta de exibição. Um objetivo de ampliação mais baixo é aceitável, particularmente se um estágio motorizado de precisão não estiver disponível. Certifique-se de que o polarizador, o analisador e o prisma estejam todos no lugar. Alternativamente, as amostras também podem ser imagens usando contraste de fase.</li>
	<li>Abra o software de imagem.</li>
	<li>Concentre o objetivo na área entre as três explantas.</li>
	<li>Salve as coordenadas (x, y e z) deste local no software de imagem.</li>
	<li>Para imaginar toda a área entre e ao redor das explants selecione a opção de adquirir uma imagem grande e especifique o tamanho da área. Isso permitirá a aquisição de múltiplas imagens ao redor da área especificada e criará uma imagem de ladrilho que representa uma região maior da amostra.</li>
	<li>Defina o tempo de exposição e a intensidade da luz aos menores valores possíveis para evitar a morte celular causada pela fonoxoxicidade, ao mesmo tempo em que fornece resolução suficiente para discriminar entre células, microesferas e fibras fibrinas.</li>
</ol>6. Rastreamento de tensão
Para obter detalhes e instruções sobre o software de rastreamento de tensão(Figura 2) usado consulte Raghupathy et al. 21 O algoritmo é um código MATLAB personalizado que pode ser baixado a partir de <a href="http://www.license.umn.edu/default.aspx">http://www.license.umn.edu/default.aspx</a>. Observe que as imagens DIC geralmente têm textura suficiente para o rastreamento de tensão. As microesferas estão incluídas para servir como uma verificação nos campos de tensão calculados. Se o rastreamento de tensão for feito, é fundamental que as imagens obtidas sejam tiradas exatamente no mesmo local para que as imagens sejam registradas. Imagens não registradas produzirão cepas espúrias.

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Nenhum conflito de interesses declarado.

Este protocolo foi desenvolvido com o objetivo de observar e quantificar a mecânica envolvida na remodelação do ECM mediado por células. Tais processos estão por trás de uma série de fenômenos biológicos e têm implicações importantes para os tecidos de engenharia2,22, reduzindo a cicatriz1,23, e entendendo a remodelação do tecido patológico12,24. O uso de microscopia DIC de lapso de tempo permite resolver e quantificar o deslocamento e o alinhamento das fibras fibrinas que ...

Uma ferramenta importante para estudar as interações entre células e matrizes é o gel de colágeno povoadocelular 1,2. O gel fornece um ambiente 3D mais próximo do caráter in vivo do tecido e mais adequado para entender o comportamento celular do que é oferecido pelas culturas 2D tradicionais3. Estudos iniciais nos quais os fibroblastos foram distribuídos de forma homogênea dentro de um gel de colágeno descobriram que as células consolidam rapidamente as fibras de colágeno e compactam o gel4,5. Os fibroblastos contratuais em géis flutuantes livres então transitam para um estado quiescente logo após o gel ter atingido totalmente a compactação1,6,7. Os fibroblastos em géis que são limitados nos limites permanecem em estado ativo e sintético8 e geram alinhamento de fibras de forma dependente da geometria do gel e restrições externas5,9. As diferenças na atividade celular parecem ser resultado da tensão interna (ou falta dela) que se desenvolve à medida que as células exercem forças de tração através de integrins nas fibras de colágeno no gel.
Uma variante dessa técnica envolve colocar explantas de fibroblastos (ou seja, aglomerados de células) a uma distância entre um gel de colágeno e observar interações de matriz celular e o desenvolvimento gradual do alinhamento de fibras entre as explantas (às vezes chamadas de correias ligamentares)10-12. A principal vantagem do sistema de explant é que ele permite que se organize as células em padrões geométricos simples, o que torna mais fácil visualizar e sondar os mecanismos subjacentes ao realinhamento de fibras orientados por células. Esses padrões de alinhamento — que dependem principalmente da interação entre as forças de tração celular, distribuição espacial celular, geometria do gel e as restrições mecânicas do gel — são importantes de entender porque desempenham um papel central na organização global de tecidos, função mecânica e ambiente mecânico local13.
No campo da engenharia de tecidos, uma estratégia para a produção de tecidos mecanicamente funcionais e projetados envolve o controle do padrão de alinhamento de fibras que se desenvolve a partir da compactação celular para que o tecido projetado possua alinhamento de fibras que imita o do tecido nativo14,15. Acredita-se que tal alinhamento seja necessário para que os tecidos projetados repliquem o complexo comportamento mecânico dos tecidos nativos. Uma modificação dessa estratégia é substituir o gel de colágeno por um gel de fibrina16. O gel de fibrina desenvolve um padrão de alinhamento semelhante ao de um gel de colágeno durante a compactação. Com o tempo, a fibrina é degradada e substituída por ECM sintetizado por células que segue o padrão inicial de alinhamento da fibra fibrina. A construção projetada resultante melhorou significativamente as propriedades mecânicas em comparação com as construções derivadas de gel de colágeno17.
O processo de alinhamento e os eventos subsequentes de remodelação em géis de fibrina prosseguem de forma complicada e mal compreendida. Para caracterizar melhor essas interações e seu efeito sobre o comportamento celular e a remodelação do ECM, desenvolvemos um procedimento baseado no método de explant. Neste método, explantas de fibroblasto são posicionadas em um gel de fibrina em diferentes padrões geométricos. Os géis são mantidos em um bioreator18controlado pelo microscópio, controlado pelo microscópio, e o processo de compactação e realinhamento de fibras é monitorado com microscopia de contraste diferencial de interferência de lapso de tempo (DIC). Os campos de deslocamento são quantificados com algoritmos personalizados. Os dados obtidos a partir desses experimentos têm amplas implicações para uma série de processos, incluindo a otimização de estratégias de engenharia tecidual, a melhoria da cicatrização de feridas e o tratamento da remodelação patológica do tecido.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invitrogen</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lonza</td>
			<td>DE14-701F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Probes</td>
			<td>F8858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher-Scientific</td>
			<td>SS264-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A3428-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Biotense Bioreactor</td>
			<td style="width:85px;">ADMET</td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti-Eclipe Microscope</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Bottom Petri Dish</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Top Petri Dish</td>
			<td>MatTek</td>
			<td>P35GTOP-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Luer fittings, PVC tubing with Luer ends</td>
			<td>Cole-Parmer</td>
			<td>30600-65</td>
		</tr>
	</tbody>
</table>

observing and quantifying fibroblast-mediated fibrin gel compaction

Células embutidas em géis de colágeno e fibrina ligam e exercem forças de tração sobre as fibras do gel. Essas forças podem levar à reorganização local e global e ao realinhamento da microestrutura de gel. Esse processo prossegue de forma complexa e dependente, em parte, da interação entre a localização das células, a geometria do gel e as restrições mecânicas do gel. Para entender melhor como essas variáveis produzem padrões globais de alinhamento de fibras, usamos microscopia de contraste diferencial de impacto de lapso de tempo (DIC) juntamente com um bioreator ambientalmente controlado para observar o processo de compactação entre explantas geométricas espaçadas (clusters de fibroblastos). As imagens são então analisadas com um algoritmo de processamento de imagem personalizado para obter mapas da cepa. As informações obtidas a partir desta técnica podem ser usadas para sondar a mecanobiologia de várias interações de matriz celular, o que tem implicações importantes para a compreensão de processos em aplicações de cicatrização de feridas, desenvolvimento de doenças e engenharia de tecidos.

A remodelagem tecidual é um processo complexo que é impulsionado em parte por interações físicas recíprocas entre as células e a matriz circundante. As células reorganizam as fibras circundantes e geram tensão na rede de fibras. O alinhamento das fibras e do ambiente mecânico, por sua vez, controla o comportamento celular, de modo que tanto as células quanto a matriz globalmente se reorganizam para produzir tecido remodelado. Neste experimento, as células das explanações, inicialmente redondas em morfologia...

Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Foram utilizadas técnicas de microscopia e processamento de imagem com lapso de tempo para observar e analisar a compactação de gel mediada por fibroblasto e o realinhamento da fibra fibrina em um bioreator ambientalmente controlado durante um período de 48 horas.

Observando e quantificando compactação de gel de fibrina mediada por fibroblasto

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

5.9K visualizações.  University of Iowa. Foram utilizadas técnicas de microscopia e processamento de imagem com lapso de tempo para observar e analisar a compactação de gel mediada por fibroblasto e o realinhamento da fibra fibrina em um bioreator ambientalmente controlado durante um período de 48 horas.

Vídeo: Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Preparação de Matrizes Reclamação para Quantificar contração celular

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Bioengenharia

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 ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

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 inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

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 ...

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

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

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

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

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

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Caracterização de leucócito-rico em plaquetas de fibrina, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formando polymersomes de tamanho gigante usando hidratação Gel-assistida

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Uma nova técnica para gerar e Observing Quimioluminescência em um ambiente Biológica

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Um Modelo de Câmara Microfluidic Fluxo de transfusão de plaquetas e Hemostasia Medidas deposição de plaquetas e formação de fibrina em tempo real

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

Gel-seq: Um método para a preparação de biblioteca simultânea de sequenciamento de DNA e RNA usando matrizes de hidrogel

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...