Este trabalho foi realizado utilizando as instalações do Departamento de Engenharia Biomédica da Universidade Internacional da Flórida.

1. Setup Electrospinning Collector <ol><li> Corte o papel alumínio em uma peça retangular. </li><li> Dobre o pedaço retangular em uma tira retangular, e anexá-lo perpendicular a uma placa de metal plana com fita adesiva (Figura 1). Nota: O tamanho da esteira de fibras dependem do comprimento e da largura da tira. Assim, as dimensões da tira pode ser ajustada conforme necessário. </li></ol> 2. Solução polimérica Preparação <ol><li> Mistura de glicerol e ácido dodecanóico (ADD) em razão molar de 1:1, num copo de 120 ° C durante 100 horas para se obter polímero de DGP. </li><li> Dissolve-se o poli (óxido de etileno) (PEO) e de gelatina, em etanol a 65% com uma proporção de peso de 1.5:3:95.5 em um tubo de 15 ml, apertar a tampa e aquecer a mistura num forno a 60 ° C durante 1 hora com agitação até que se torna uma solução homogénea (solução basal). </li><li> Para electrospinning, misture o polímero PGD ea solução basal em 04:06 de peso. Nota: concentração PGD tem um big efeito sobre o diâmetro da fibra. 30% -50% por cento PGD é aceitável para a produção de fibras mais do que 5 cm com diâmetros de fibra de aumento. </li><li> Adicionar 0,1% de riboflavina para a solução polimérica e misturar bem. </li></ol> 3. Electrospinning <ol><li> Alimente a solução polimérica em uma seringa de 5 ml de série com um 18 G embotada agulha de aço inoxidável. </li><li> Insira a seringa em uma bomba de seringa. </li><li> Ligue o cabo ligado à terra de uma fonte de alimentação de alta tensão para a placa de metal e o chumbo carregada positivamente para a agulha. </li><li> Ajustar a distância entre a agulha e a tira de folha de alumínio de 15 cm. </li><li> Coloque a bomba de seringa em um ângulo de aproximadamente 15 ° com a horizontal para impedir a agregação das fibras na parte da frente da faixa. </li><li> Ligar a bomba de seringa e ajustar a taxa de fluxo da bomba de 0,6 mL / hora. </li><li> Ligue a fonte de alimentação de alta tensão e ajustar a tensão de operação de 14,6 kV. </li></ol> 4.Processamento de fibras <ol><li> Após a recolha está completa, expor a esteira de fibra para a luz UV, durante 60 minutos para a ligação cruzada. </li><li> Transferir a esteira de fibras a partir da tira de folha de alumínio de uma placa de Petri de 100 milímetros, e expô-la à luz UV durante mais 20 min para a esterilização. </li><li> Em uma cabine de segurança biológica, cortar a manta de fibra em peças redondas do mesmo tamanho com uma lâmina de bisturi e colocar os pedaços em uma placa de 24 poços. </li><li> Para o tratamento das células de pré-sementeira, imergir amostras de fibras em 1 ml de tampão fosfato salino (PBS) e incuba-se a 37 ° C durante a noite. </li><li> No dia seguinte, cuidadosamente aspirado PBS, adicionar 1 ml de meio de diferenciação (DMEM/F12, N2, e FGF2) (ver receita na tabela Materiais) a cada poço e incuba-se a 37 ° C durante 3 hr. </li><li> Meio de diferenciação aspirado cuidadosamente, adicionar 0,2 mL de matrigel a cada amostra de fibra e incubar a 37 ° C durante 30 min. Nota: A laminina também pode ser utilizado para o revestimento de fibra. Adicionar 0,2 mL de 20 ug / ml de lamininapara a fibra e incubar à temperatura ambiente durante 3 hr. </li><li> Com cuidado, retire o excesso de matrigel de cada poço. Lavar com 2 ml de meio de diferenciação de uma vez. Nota: As amostras de fibras estão prontos para a cultura de células. </li></ol> 5. Sementeira celular em Fibras <ol><li> Adicionar 1 ml de Accutase para o prato de cultura de células MES e incubar durante 10 min a 37 ° C. </li><li> Após 10 min, as células MES são separadas do prato de cultura. Adicionar 4 ml de meio de diferenciação para a placa. Recolher as células MES flutuantes em um tubo de 15 ml e as células para cima e para baixo de pipeta para quebrar colónias (cerca de 15x). </li><li> Centrifugar a 400 xg durante 5 minutos e re-suspender as células em 4 ml de meio de diferenciação. </li><li> Contar as células usando um hemocitômetro. Transferir 200 ul da suspensão de células para um tubo de 15 ml e diluir 10x com meio de diferenciação. Transferir 15 uL da suspensão de células diluída a uma câmara na hemocitómetro com uma lamela no lugar. Contaras células no quadrado do centro a 1 mm e as quatro esquinas do hemocitómetro sob o microscópio. Nota: celular por ml = a contagem média por metro quadrado x fator de diluição x 10 4 </li><li> Colocar aproximadamente 5 x 10 4 células MES em cada poço. Gota lentamente a suspensão de células para o meio das amostras de fibras, em vez de correr para fora do lado do poço, para evitar que a solução de se drenar o tapete de fibra. </li><li> Adicionar 1 ml de meio de diferenciação em cada poço, e manter a placa numa incubadora a 37 ° C e 5% de CO2 para permitir que as células de fixação, o crescimento e diferenciação. </li><li> Aspirar o meio antigo em cada poço e substitua com 1 ml de meio de diferenciação fresco todos os dias. </li></ol> 6. Viabilidade celular <ol><li> Aspirar o meio antigo em cada poço. </li><li> Misture 1/10 de volume de Resazurina reagente de fluorescência com o meio de cultura e adicionar 1 ml para cada poço. </li><li> IncubComeram a 37 ° C e 5% de CO 2 durante 4 h, protegido da luz directa. </li><li> Depois de 4 horas, transferir 100 mL 3x do reagente de cada poço de uma placa de 96 poços, em seguida, as amostras estão prontas para ser medido em um espectrofotômetro de fluorescência usando configurações de filtro de 560EX nm/590EM. </li></ol> 7. PCR em Tempo Real <ol><li> Após 14 dias de cultura, adicionar tampão de lise às amostras e isolar o RNA total a partir das células nas amostras de fibras. </li><li> Use 4 ul de ARN total, para sintetizar ADNc em 20 uL de escalas de reacção de transcrição reversa. </li><li> Preparar a mistura de reacção de PCR em cada um dos tubos óptica: 10 ul Master Mix (2x), 0,2 ul de corante, 8 mL de água livre de nuclease, 1 ul de cDNA, e um iniciador pi (iniciadores utilizados neste estudo estão listados na Tabela 1). </li><li> Configure o programa de PCR: a. 95 ° C 02:20 min, um ciclo de b. 95 ° C 3 seg → 60 ° C 1 min, 40 ciclo c. 95 ° C 15 seg, um ciclo d. 60 ° C 1min, um ciclo de e. 95 ° C 15 seg, 1 ciclo. Escolha forma de C T comparativo para determinar os níveis de expressão gênica relativa. </li><li> Após PCR for concluído, analisar os resultados em tempo real PCR com software StepOne e exportar os resultados para o Excel. </li></ol>

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P., Sell, S. A., Boland, E. D., Simpson, D. G., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+technology:+designing+the+next+generation+of+tissue+engineering+scaffolds.">Nanofiber technology: designing the next generation of tissue engineering scaffolds.</a> Advanced drug delivery reviews. 59, 1413-1433 (2007).</li><li>Li, W. -. J., Mauck, R. L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds:+production,+characterization,+and+applications+for+tissue+engineering+and+drug+delivery.">Electrospun nanofibrous scaffolds: production, characterization, and applications for tissue engineering and drug delivery.</a> Journal of Biomedical Nanotechnology. 1, 259-275 (2005).</li><li>Pham, Q. P., Sharma, U., Mikos, A. 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As limitações de coletores simples ou as complexidades de colecionadores que são usados ​​atualmente para eletrofiação aumentar a restrição de se obter o comprimento desejado e tamanho do tapete de fibra para algumas aplicações rotativas. Além disso, a transferência de fibras do coletor de terra para o prato de cultura ou outros substratos é um desafio 5. Neste relatório, um coletor recém-concebido, feito, simplesmente anexando uma tira de folha de alumínio para o coletor de castigo, foi ca...

Eletrofiação é um dos métodos de processamento eficazes para produzir suportes de fibra de tamanho micro-a-nanómetro. O princípio básico de electrospinning envolve um cone de Taylor de uma solução que é realizada ao nível do orifício de uma agulha através da aplicação de alta tensão entre a ponta da agulha e de um colector ligado à terra. Quando a repulsão electrostática na solução vence a tensão superficial, um jacto de fluido carregado é ejectado para fora da ponta da agulha, se desloca através do ar com a evaporação do solvente, e é, finalmente depositada sobre o colector ligado à terra. A bomba de seringa fornece um fluxo contínuo de uma solução que emerge da fieira e, portanto, múltiplas cópias das fibras electrospun pode ser fabricado dentro de um curto período de tempo. Durante o curso de deixar a fieira de chegar ao colector, o jacto carregada sofrer estiramento e batimento de acordo com uma série de parâmetros, que incluem a viscosidade e tensão superficial da solução polimérica, os electrostatic força na solução, ea interação do campo elétrico externo, etc 2. No processo de electrospinning, um colector serve como um substrato condutor em que as fibras de micro-a-nanométrica pode ser depositado. Neste estudo, um novo tipo de colector de fibras foi concebido para obter mantas de fibra com a dimensão desejada (comprimento x largura). Tradicionalmente, a folha de alumínio é usado como um colector, mas é difícil de transferir as fibras a partir da superfície plana a um outro substrato. A dificuldade de colher um tapete de fibra intacta de um colecionador tradicional deveu-se principalmente ao fato de que as fibras electrospun anexar fortemente à superfície do coletor. Portanto, nós modificamos o coletor dobrando um pedaço de papel alumínio em uma tira retangular e anexá-lo perpendicular a uma placa de metal plana. As fibras são electrospun esticado em toda a área entre a ponta da tira e a placa de metal, que pode ser facilmente transferida para outro substrate. Interesse em polímeros elastoméricos termicamente reticuladas está crescendo rapidamente por causa do trabalho pioneiro do grupo de Robert Langer, que introduziu poli (sebaçato glicerol) (PGS), um poliéster que é análoga à borracha vulcanizada, em 2002 3. Semelhante ao PGS, temos desenvolvido com sucesso poli (glicerol-dodecanoato) (PGD) por condensação térmica do glicerol e ácido dodecanodioíco e demonstrou a sua propriedade única memória de forma 1. Ao contrário rígida poli materiais sintéticos (hidroxil-butirato) ou poli (L-lactido) (módulos de Young de 250 MPa e 660 MPa, respectivamente), o PGD exibe propriedade elastomérica como a borracha, com um módulo de Young de 1,08 MPa, quando a temperatura estiver acima de 37 ° C, que é um jogo perto do nervo periférico in-situ (0,45 MPa). Além disso, o PGD é biodegradável e o tempo de degradação, podem ser afinadas variando a proporção de glicerol e ácido dodecanóico. Ácido dodecanodioíco é um sub de doze carbonoposição com dois grupos carboxílicos terminais, HOOC (CH2) 10 COOH. Ácidos dicarboxílicos pares numeradas como ácido sebácico e o ácido dodecanodióico pode ser metabolizado a acetil-CoA e introduzir o ácido tricarboxílico (TCA) / ciclo (ácido cítrico). O produto metabólico de ácidos dicarboxílicos, succinil-CoA, é um precursor gluconeogenetic e intermédia do ciclo de TCA 4. Assim, alguns estudos sugerem que poderiam ser utilizados como um substrato de combustível alternativo para nutrição entérica e parentérica, especialmente nas condições patológicas. Além disso, o PGD exibe memória de forma única por causa da sua temperatura de transição de vidro é de 31 ° C, portanto, apresenta propriedades mecânicas diferentes, à temperatura ambiente e à temperatura do corpo. Em suma, a PGD é biodegradável, biocompatível, exibindo propriedades elásticas únicas com propriedades mecânicas semelhantes aos tecidos nervosos; por conseguinte, é um material adequado para aplicações de engenharia de tecidos nervosos. Neste protocolo, o electrospunfibras longas que abrangem uma grande área de depósito foram fabricados através do coletor recém-projetado do PGD. Os andaimes de fibras pode suportar o crescimento e diferenciação de células-tronco pluripotentes.

<table><tbody><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7757</td><td></td></tr><tr><td>Dodecanedioic acid</td><td>Sigma-Aldrich</td><td>D1009</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>D1890</td><td></td></tr><tr><td>Poly(ethylene oxide) (PEO)</td><td>Sigma-Aldrich</td><td>182028</td><td></td></tr><tr><td>Riboflavin</td><td>Sigma-Aldrich</td><td>132350250</td><td>0.10%</td></tr><tr><td>Mouse embryonic stem cells</td><td>GlobalStem</td><td>GSC-5002</td><td></td></tr><tr><td>Matrigel</td><td>Becton Dickinson</td><td>356234</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo Scientific</td><td>SH30272.02</td><td></td></tr><tr><td>N2 supplement</td><td>Invitrogen</td><td>17502048</td><td>1%</td></tr><tr><td>FGF2</td><td>Stemgent</td><td>03-0002</td><td>10 ng/ml</td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>A11105-01</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Invitrogen</td><td>10010-031</td><td></td></tr><tr><td>Resazurin fluorescence dye</td><td>Sigma-Aldrich</td><td>62758-13-8</td><td></td></tr><tr><td>SV Total RNA Isolation System</td><td>Promega</td><td>Z3100</td><td></td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5000</td><td></td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6001</td><td></td></tr><tr><td>Syringe pump&amp;nbsp;</td><td>Fisher scientific</td><td>14-831-200</td><td></td></tr><tr><td>High voltage power source&amp;nbsp;</td><td>Spellman High Voltage Electronics Corporation</td><td></td><td>SL30</td></tr><tr><td>UV light</td><td>Philips</td><td>308643</td><td>15W/G15T8</td></tr><tr><td>Synergy HT Multi-Mode Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Perkin Elmer GeneAmp PCR System 9600</td><td>Perkin Elmer</td><td>8488</td><td></td></tr><tr><td>StepOne Real-time PCR System</td><td>Applied Biosystems</td><td>4376357</td><td></td></tr></tbody></table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

Para aplicações de engenharia de tecidos, a preparação de scaffolds biodegradáveis ​​e biocompatíveis é a tarefa mais desejável, mas desafiador. Entre os vários métodos de fabricação, electrospinning é o mais atractivo, devido à sua simplicidade e versatilidade. Além disso, nanofibras electrospun imitar o tamanho da matriz extracelular natural, assegurando um apoio adicional para a sobrevivência e crescimento das células. Este estudo mostrou a viabilidade da fabricação de fibras longas, abrangendo uma área maior depósito de um polímero biodegradável e biocompatível romance chamado poli (glicerol-dodecanoato) (PGD) 1, usando um coletor recém-projetado para electrospinning. PGD ​​exibe propriedades elásticas originais com propriedades mecânicas semelhantes às dos tecidos nervosos, assim, ele é adequado para aplicações de engenharia de tecidos neurais. A síntese e fabricação set-up para fazer materiais fibrosos andaimes era simples, altamente reprodutível e barato. Em biocompatibilidadeteste, as células derivadas de células estaminais embrionárias de ratinho podia aderir e crescer nas fibras PGD electrospun. Em resumo, este protocolo fornecido um método de fabricação versátil para fabricação de fibras PGD electrospun para suportar o crescimento de células da linhagem neurais derivadas do rato de células-tronco embrionárias.

Os principais componentes do electrospinning são mostrados na Figura 1. Uma esteira de fibras de tamanho grande foi tipicamente obtida através da tira perpendicularmente ligada folha de alumínio e uma chapa de metal plana. Figura 2 mostra o desenho do colector e o tapete de fibra de electrospinning. A largura e o comprimento pode ser ajustado para diferentes aplicações. O comprimento da fibra feito com polímeros de PGD e mistura solução basal é de até 10 cm. A morfologia das f...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Síntese e fabricação de fibras longas electrospun abrangendo uma área maior depósito através de um coletor recém-concebido a partir de um polímero biodegradável romance chamado poli (glicerol-dodecanoato) (PGD) foi relatado. As fibras eram capazes de suportar o crescimento de células derivadas de células-tronco pluripotentes.

Electrospun fibrosa Andaimes de Poli (glicerol-dodecanedioate) para Engenharia Neural tecidos de rato as células estaminais embrionárias

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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Research

JoVE Journal

Bioengineering

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

10.8K visualizações.  Florida International University.  Síntese e fabricação de fibras longas electrospun abrangendo uma área maior depósito através de um coletor recém-concebido a partir de um polímero biodegradável romance chamado poli (glicerol-dodecanoato) (PGD) foi relatado. As fibras eram capazes de suportar o crescimento de células derivadas de células-tronco pluripotentes. 

Vídeo: Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

A Cultura da Motor Primário e neurônios sensoriais no Media Definidas em electrospun Poly-L-ácido lático nanofibras Andaimes

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Neurociência

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 ...

Bioengenharia

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Electrospinning Fator de Crescimento Releasing Microesferas em fibrosa Andaimes

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Electrospun nanofibras Andaimes com gradações de Organização Fiber

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

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 ...

Biologia

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Eletricamente condutivo Andaime modular e entregar as células-tronco

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Redes neuronais modificadas por micro-tecidos tridimensionais com inspiração anatômica para a reconstrução, modulação e modelagem do sistema nervoso

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.