Este trabalho foi financiado por um americano Fellowship Heart Association Predoctoral para JLI e National Institutes of Heath conceder R01AR056624.

A. Único Polymer Electrospinning <ol><li> Antes de preparar a solução de eletrofiação, faça uma solução 0,5% em peso do fotoiniciador, IRGACURE 2959 (I2959), em água deionizada, dissolvendo a 37 ° C por vários dias. Este passo não é necessário se um polímero fotorreativas não está sendo usado. </li><li> Combine ácido hialurônico methacrylated (MEHA, ver Burdick et al. Para a síntese), poli (óxido de etileno) (PEO, 900 kDa), e I2959 em água deionizada para preparar uma solução com uma concentração final de 2% em peso MEHA, 3% em peso PEO, e 0,05% em peso I2959. Use um vórtice para misturar a solução até que fique claro. O tipo de polímero e concentração, bem como o solvente usado pode ser modificado nesta etapa, dependendo das propriedades desejadas andaime. </li><li> Transferir a solução para uma seringa e anexar uma calibre 18, seis polegadas de comprimento, agulha de ponta cega até o fim. </li><li> Bomba de programa de uma seringa para ejetar a uma taxa de 1,2 mL / hr. Inserir a seringa ea agulha para o dispositivo. </li><li> Anexar a liderança fundamentada de uma fonte de alta tensão para o aparelho de coleta. Para coletar aleatoriamente distribuídos fibras usar uma placa de metal lisa, ou usar um mandril girando a ~ 10 m / s para coletar fibras alinhadas. Anexar a liderança com carga positiva para a agulha. Veja a Figura 1 para um esquema do aparelho eletrofiação. </li><li> Ajustar a agulha ou dispositivo de recolha, de modo que há uma distância de 15 cm entre os dois. </li><li> Iniciar o fluxo da bomba de seringa. Quando o fluido é visualizada na ponta da agulha, ligue a fonte de alimentação e definir a tensão de 22 kV. </li><li> Após a coleta é completo (a partir de vários minutos para filmes finos, com até 24 horas para um mais grosso tapete), remova o andaime do aparelho de coleta e armazená-lo sob vácuo durante a noite para garantir a remoção completa do solvente. </li><li> Visualizar a morfologia da fibra através de microscopia eletrônica de varredura (MEV), representante de um andaime é mostrado na Figura 1 para ambas as estruturas alinhadas e não-alinhados. </li></ol> Nota: O caudal da amostra, a distância para o dispositivo de recolha e de tensão dependem da combinação de polímero e solvente e devem ser otimizados para cada sistema, normalmente por meio da observação da morfologia andaime com SEM. B. Photocrosslinking e Photopatterning <ol><li> Cortar amostras 5 milímetros X 5mm do tapete andaime. </li><li> Prepare-se para crosslinking, colocando cada um andaime em uma folha de lâmina de vidro coberta, colocando a fotomáscara diretamente no cadafalso, cobrindo com uma lâmina de vidro limpa e clipping ambas as extremidades com grampos da pasta. Nota: quatro amostras pode ser feito de uma só vez, e uma transparência sem um padrão pode ser usado para crosslinking se um padrão não é desejada. </li><li> Purge andaime montado em uma câmara de nitrogênio. É importante manter a construir livre de oxigênio, que pode inibir a reticulação. </li><li> Configuração local andaime na câmara de nitrogênio em ~ 10 mW / cm 2 luz de 365 nm com um adaptador de colimação por 5 minutos. Ver esquema na Figura 2. </li><li> Retire cada scaffold e coloque em um prato de 12 poços. </li><li> Adicionar 2 mL de água deionizada em cada poço, a placa de parafilme para evitar a evaporação da água e coloque a 37 ° C por 24 horas. Troque a água três vezes. </li><li> Visualize formação de poros em microscópio de luz (ver exemplo na Figura 2). Os andaimes estão agora prontos para uso. </li></ol> Nota: Photocrosslinking não é necessário para muitos tipos de polímeros, no entanto photopatterning só pode ser usado com polímeros fotorreativas. C. Dupla Polymer Electrospinning com Fluorescente Visualization Fiber <ol><li> Prepare uma solução 5% em peso de PEO (200 kDa) em etanol 90%. Mexa a 700 rpm a 50 ° C por pelo menos 2 horas antes da eletrofiação. </li><li> Transferir a solução PEO a uma seringa e adicionar DAPI para uma concentração final de 10 concentração mg / mL na solução eletrofiação. Enrole a seringa na folha de alumínio para proteger da luz. </li><li> Prepare a mesma solução descrita na etapa A2, com exceção também adicionar methacryloxyethyl thiocarbamoyl rodamina B (MeRho, 683,24 g / mol) para uma concentração final de 25 mM na solução eletrofiação. Enrole a seringa na folha de alumínio para proteger da luz. </li><li> Com cuidado, use fita adesiva para garantir lamínulas de vidro methacrylated (ver Khademhosseini et al.) Para a superfície do mandril. Nota: as fibras ficarão amarrados em lamínulas de vidro methacrylated. </li><li> Conecte uma extremidade de um pedaço 5 mm de comprimento de tubo de silicone com anexos luer lock para uma das seringas e anexar a outra extremidade a uma agulha. Inserir a seringa na bomba de seringa e colocar a agulha através do orifício na fazendeiro. Repita com a outra seringa e fazendeiro no lado oposto do mandril. Os lavradores traduzir o llength do mandril e são usados ​​para garantir uma distribuição igual dos dois polímeros no andaime resultante. Ver esquema na Figura 3. </li><li> Ajustar os parâmetros descritos na secção A forma adequada de acordo com a Tabela 1. Garantir que as pontas de agulha são centradas no mandril. Tabela 1. Dupla Polymer Parâmetros Electrospinning <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Seringa </td><td> Fazendeiro a ponta da agulha (cm) </td><td> Dica agulha para Mandril (cm) </td><td> Vazão (mL / hr) </td><td> Tensão aplicada (kV) </td></tr><tr><td> MEHA </td><td> 6 </td><td> 15 </td><td> 1,2 </td><td> 22 </td></tr><tr><td> PEO </td><td> 6 </td><td> 10 </td><td> 1,2 </td><td> 15 </td></tr></tbody></table> </li><li> Depois que tudo estiver alinhado corretamente, desligue as luzes e ligar o mandril e as bombas de seringa. Remova o papel alumínio do seringas. Quando o fluido é visível nas pontas de ambas as agulhas, simultaneamente ligar as fontes de alimentação e conecte os fazendeiros. </li><li> Quando a coleta for concluída, desligue a fontes de alimentação e desconecte o mandril e lavradores. Remova cuidadosamente as lamelas e fita usando uma lâmina de barbear. </li><li> Visualize as fibras utilizando um microscópio de fluorescência equipado com filtros para rodamina e DAPI. Veja o exemplo na Figura 3. Nota: fibras fluorescentes são mais claros para ver quando electrospun por curtos períodos de tempo (&lt;3 minutos), no entanto, este processo pode ser prorrogado por uma quantidade ilimitada de tempo para criar construções mais espesso, especialmente sem o uso do lamínulas de vidro . </li></ol> Células Seeding D. em Andaimes <ol><li> Coloque cada lamela com electrospun polímero anexado em um indivíduo bem de uma placa de tamanho adequado também. Nota: interações celulares com as fibras são facilmente visíveis por eletrofiação em lamínulas de vidro methacrylated. Além disso, MeRho pode ser novamente utilizado para a visualização de fibra. </li><li> Incubar os andaimes em PBS durante a noite para garantir a remoção completa do solvente e qualquer lixiviável possíveis subprodutos. </li><li> Remover o PBS dos poços. </li><li> Para esterilizar os andaimes, colocá-los sob uma lâmpada germicida de uma capela de fluxo laminar por 30 minutos. Se um andaime completo está sendo semeada, flip do andaime e coloque sob uma lâmpada germicida por mais 30 minutos. </li><li> Realizar cultura de células padrão e preparar uma suspensão de células concentradas na densidade de célula desejada (por exemplo, 6.000 células cm -2). Por exemplo, usar um 100 mL de suspensão celular por um 22 mm X 22 mm lamela. Lugar na incubadora por 1 hora. </li><li> Adicionar a quantidade adequada de meios de cultura de células em cada poço. Coloque o andaimes de volta para a incubadora. </li><li> Mancha as células usando um kit comercialmente disponível Live / Dead. Nota: outros tipos de coloração de células, tais como DAPI (núcleos celulares) e phalloidin fluorescente etiquetado (fibras de actina estresse) pode ser aplicado para visualizar as células. </li><li> Visualize as células e fibras usando um microscópio de fluorescência equipado com filtros para TRITC e FITC. Veja o exemplo na Figura 4. </li></ol> Resultados representativos: <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. Esquemático ilustrando a configuração do dispositivo para não-alinhados formação andaime (superior) e formação alinhada andaime (parte inferior). Exemplo, imagens de microscopia eletrônica de varredura de cada tipo de andaime são mostrados. Barra de escala = 5m. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="Figura 2" /> Figura 2. Esquemática do método de padronização. Padrões são formadas pela colocação de um fotomáscara entre a fonte de luz e andaimes durante photocrosslinking e depois lavar polímero não reagiu. Fotomáscara e SEM imagem de andaimes após a formação dos poros e liofilização. Barra de escala = 100 mm. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">clique aqui</a> para ver uma versão ampliada da figura 2. <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="Figura 3" border="0" /> Figura 3 Esquema da configuração electrospinning multi-polímero e parâmetros de processamento necessários, bem como uma imagem representativa fluorescentes de uma mistura de duas populações de fibra (exemplo: vermelho e azul MEHA PEO). Qual foram simultaneamente electrospun para formar um andaime multi-polímero . Barra de escala = 100 mm. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">clique aqui</a> para ver um maior version da figura 3. <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="Figura 4" /> Figura 4. Um exemplo de imagem coloração Live / Dead of células-tronco mesenquimais e suas interações com as fibras electrospun. Barra de escala = 100 mm. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">clique aqui</a> para ver uma versão ampliada da figura 4.

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

Eletrofiação foi usado para preparar andaimes fibroso a partir de polímeros. Photocrosslinkable andaimes à base de ácido hialurônico foram usadas como um exemplo ilustrativo, onde a exposição à luz é necessário para a reticulação. Com o uso de macromers reativa, como MEHA, canais que já demonstrou melhor distribuição celular foram incorporados ao andaimes com o uso de uma máscara durante photocrosslinking andaimes para formar macro e micro-porosa. Além disso, dois polímeros distintos foram simultaneam...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

Como o campo da engenharia de tecidos evolui, há uma enorme demanda para a produção de materiais mais adequados e técnicas de processamento, a fim de atender aos requisitos (por exemplo, mecânica e vascularização) de órgãos e tecidos mais intrincados. Eletrofiação é uma técnica popular para criar andaimes fibroso que imitam a arquitetura ea escala de tamanho da matriz extracelular nativa. Estes andaimes fibroso também são úteis como substratos de cultura de células já que as fibras podem ser usadas para orientar o comportamento celular, incluindo a diferenciação de células-tronco (ver extensas revisões por Mauck<em&gt; Et al.</em&gt; E Sill<em&gt; Et al.</em&gt; Para mais informações). Neste artigo, descrevemos o processo geral de eletrofiação polímeros e como um exemplo, um ácido hialurônico electrospin reativos capazes de reticulação com exposição à luz (ver Ifkovits<em&gt; Et al.</em&gt; Para uma revisão sobre os materiais photocrosslinkable). Também introduzimos capacidades de processamento, tais como: photopatterning e multi-polímero de formação de andaime. Photopatterning pode ser usado para criar andaimes com canais e multi-escala de porosidade para aumentar a infiltração celular e distribuição do tecido. Multi-polímero de andaimes são úteis para ajustar melhor as propriedades (mecânica e degradação) de um andaime, incluindo a porosidade sob medida para infiltração celular. Além disso, estas técnicas podem ser estendidas para incluir uma grande variedade de polímeros e macromers reativa para criar andaimes complexos que fornecem as pistas necessárias para o desenvolvimento de construções de tecido bem sucedida de engenharia.

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

O processo de eletrofiação polímeros para engenharia de tecidos e de cultura de células é abordada neste artigo. Especificamente, o electrospinning de macromers fotorreativas com capacidades de processamento adicional de photopatterning e multi-polímero eletrofiação é descrito.

Eletrofiação Andaimes Polymer fibrosos para Engenharia de Tecidos e Cultura de Células

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

Research

JoVE Journal

Biology

21.4K visualizações.  University of Pennsylvania. O processo de eletrofiação polímeros para engenharia de tecidos e de cultura de células é abordada neste artigo. Especificamente, o electrospinning de macromers fotorreativas com capacidades de processamento adicional de photopatterning e multi-polímero eletrofiação é descrito.

Vídeo: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

Abordagens experimentais de Engenharia de Tecidos

Biologia

Micro-scale Engineering for Cell Biology

Micro-escala de Engenharia de Biologia Celular

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Ensaio Co Sedimentação; para a Análise da proteína de ligação a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricação de Chip porte Andaimes para tridimensional de cultivo celular

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Entrevista: biorreatores e apareceu-Modified 3D-andaimes para Investigação em Células Estaminais

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

O encapsulamento celular em Hidrogéis 3D para Engenharia de Tecidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Engenharia de Proteínas Cell-permeáveis

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Um sistema para a cultura de células epiteliais pigmentadas da íris para estudo da regeneração Lens em Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Transdução de células humanas com Polymer-complexado Lentivirus Ecotropic para reforço de Biossegurança

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Indução de hipóxia e Testes em Cultura de Células

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...