Name: engineered 3d silk-collagen-based model of polarized neural tissue
Uploaded: 2015-10-23T14:00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

O protocolo de isolamento tecido cerebral foi aprovado pelo Comitê da Universidade Tufts Animal Care Institucional e Use e está em conformidade com o National Institutes of Health Guide para o Cuidado e Uso de Animais de Laboratório (Institutional Animal Care e do Comitê Use B2011-45). 1. Silk Andaime Preparação <ol><li> Preparação de solução de seda Bombyx mori casulos como descrito anteriormente 19,20. <ol><li> Corte cada casulo em 8 pedaços iguais usando uma tesoura. Use cerca de 11 casulos para 5 g de casulos fragmentados. (15 min) </li><li> Prepare 2 L de 0,02 M Na 2 CO 3 solução e trazê-lo para ferver utilizando um prato quente. (15 min) </li><li> Pesar 5 g de casulos fragmentados e ferve-las em Na 2 CO 3 solução durante 30 min. Agita-se a seda ferver a cada dois minutos com uma espátula. Esta etapa, também chamado de-resinagem, purifica fibroína de seda a partir de proteínas hidrófilas, sericins. (30 min) </li><li&gt; Torça o fibroï¿½a à mão e lave-o em água destilada pelo menos três vezes para lavar qualquer sericina restante e produtos químicos. (5 min) </li><li> Coloque a fibroína molhado numa placa de Petri e secar o extracto de fibroína na câmara de fluxo S / N. </li><li> No dia seguinte, pesar a massa seca fibroína e colocar a fibroína numa proveta de vidro. (15 min) </li><li> A fim de dissolver a fibroína em 9,3 M solução de LiBr, calcular o volume necessário (em ml) de LiBr multiplicando a massa de fibroína seco por 4. verter lentamente sobre solução de LiBr a fibroína de seda e mergulhe todas as fibras de fibroína usando uma espátula. Tapar o copo para evitar a evaporação e colocá-lo a 60 ° C durante 4 horas para permitir que as fibras se dissolva. (15 min) </li><li> Usando a seringa, recolher a solução fibroï¿½a do copo e carregá-lo para os MWCO 3.500 cassetes de diálise. Executar a diálise contra água destilada durante 48 horas. Troque a água a cada duas horas. </li><li> Usando a seringa, recolher a solução de fibroínaas cassetes em tubos de 50 ml de centrífuga cónicos e duas vezes a 9000 rpm (~ 12.700 xg) a 4 ° C durante 20 min. Verter o sobrenadante para um tubo fresco após cada passo de centrifugação e descarte do sedimento. (40 min) </li><li> Medir a concentração de fibroína através da estimativa do peso seco. Despeje 1 ml de solução de seda em um barco pesar. Seca-se a amostra no forno a 60 ° C durante 2 horas. Pesar a fibroína de seda seca e calcular a concentração de uma solução de fibroína de seda, multiplicando o peso obtido por 100. A concentração esperada de solução de seda é de 6-9% (w / v). </li><li> Ajustar a concentração de seda a 6% (w / v) diluindo-o em água destilada. Ponto de paragem: O líquido de fibroína de seda pode ser armazenado a 4 ° C durante até um mês em um recipiente fechado. </li></ol></li><li> Preparação de matrizes porosas a partir de solução de seda. <ol><li> Peneirar o granulado de NaCl para separar os grânulos de tamanho 500-600 um, que será utilizado nas etapas posteriores. Descartar o grânulos dimensionados abaixo de 500 um e acima de 600 um. (15 min) </li><li> Pour 30 ml de solução de seda 6% no molde de politetrafluoretileno (PTFE) (Figura 1). Delicadamente espalhar 60 g de NaCl peneirada sobre a seda. Toque no recipiente para se obter uma camada uniforme de sal. Incubar 48 horas à temperatura ambiente para polimerizar o seda. </li><li> Coloque o molde de PTFE contendo andaime no forno a 60 ° C durante 1 h para finalizar a polimerização e evapora-se o líquido restante. </li><li> Colocar o conteúdo do molde de PTFE num copo contendo 2 L de água destilada durante 48 horas para lixiviar o sal. Troque a água 2-3 vezes por dia. Remover os andaimes de esponja dos moldes quando o sal é completamente lixiviado para fora do ponto final:. As esponjas podem ser armazenadas imersas em água a 4 ° C num recipiente fechado para evitar que os suportes de desidratação. </li><li> Quando estiver pronto, cortar os andaimes com 5 milímetros de diâmetro biópsia soco. Pré-cortar os andaimes para chegar a cerca de 2 mm de altura. PuNCH ​​para fora do centro do andaime com a 2 mm de diâmetro punção para biópsia (Figura 2A). (1 h) </li><li> Autoclave os andaimes imersas em água para esterilizá-los (ciclo molhado, 121 ° C, 20 min) Ponto de paragem:. As esponjas podem ser armazenadas imersas em água a 4 ° C num recipiente fechado para evitar que os suportes de desidratação. </li><li> Antes da sementeira celular prevista, imergir os suportes na solução estéril a 0,1 mg / ml de poli-D-lisina (PDL). Incubar durante 1 hora a 37 ° C. </li><li> Lavar os andaimes 3x com solução salina tamponada com fosfato (PBS) para remover PDL não ligado. (30 min) </li></ol></li></ol> 2. Isolamento de Rato Os neurónios corticais <ol><li> Dissecar córtices do dia embrionário 18 ratos (E18) Sprague-Dawley como previamente descrito por Pacifici e Peruzzi 27 após protocolo de animais aprovado seja obtido. (2 h) </li><li> Incubar 10 córtices em 5 ml de 0,25% de tripsina com 0,3% de DNase I (a partir de pâncreas bovino) durante 20 min a 37 ° C. </li><li> Inactivar a tripsina por adição de igual volume de 1 mg / ml de proteína de soja. </li><li> Triturar os córtices usando 10 ml de pipeta Pasteur por pipetagem cima e para baixo 20 vezes, até uma suspensão única célula é gerada. Seja gentil e evitar a formação de bolhas de ar. (10 min) </li><li> Centrifugar a suspensão de células a 127 xg durante 5 min. </li><li> Re-suspender o sedimento celular em 10 ml de meio de cultura (meio Neurobasal, 1x suplemento de B27, 1x Glutamax, 1% de penicilina / estreptomicina). Contar as células. A concentração de células é de cerca de 2x10 esperado 7 / ml. (20 min) </li></ol> 3. Construa Assembleia e Cultura <ol><li> Semeadura andaime com células. <ol><li> Mova os andaimes estéreis e todos os utensílios necessários dentro de uma capa de cultura de célula. Utilizando fórceps estéreis os colocar andaimes em uma placa de 96 poços de cultura de células alocar um andaime por poço. (10 min) </li><li> Mergulhe os andaimes em meio de cultura de células para equilibrar-los antes para a célula de sementesing. Incubar durante 1 hora a 37 ° C. (10 min) </li><li> Aspirar o excesso de a forma de andaimes. </li><li> Aplicar 100 ul de suspensão de células / andaime. (10 min) </li><li> Incubar as células a 37 ° CO / N para permitir a fixação de células ao andaime. </li><li> Na manhã seguinte aspirar as células não ligadas e aplicar 200 ul / poço de meio de cultura fresco. (10 min) </li></ol></li><li> Andaime com a incorporação de matriz de colagénio. (2 h) <ol><li> Coloque 10x PBS, água, NaOH 1 N e rato cauda I colágeno no gelo. Preparar uma solução de trabalho de colagénio de acordo com as instruções do fabricante. Manter em gelo até que as construções de células semeadas está pronto (até 1 hora). </li><li> Retirar as construções de células-seda semeado da incubadora e aspirar o excesso de meio. </li><li> Utilizando fórceps estéreis os andaimes para transferir os poços vazios na placa e assim imergir cada andaime em 100 ul de solução de colagénio de 3 mg / ml. Coloque a placa de cultura de tecidos de voltana incubadora durante 30 min para permitir a polimerização do colagénio. </li><li> Adicionar 100 ul de meio de cultura de células pré-aquecido / poço. Cultura das construções durante uma semana de mudar o meio todos os dias, substituindo metade do volume do meio. </li></ol></li></ol> 4. Análise de Microscopia <ol><li> Coloração Live / mortos (1 hr) <ol><li> Preparar a solução de estoque de iodeto de propídio (PI) a 1 mg / ml (1,5 mM) em água destilada. </li><li> Preparar a solução de estoque de diacetato de fluoresceína (FDA) de 2 mg / ml em acetona. </li><li> Preparar uma solução de trabalho contendo 10 PI uM e 0,15 uM FDA diluído em PBS. Pré-aquecer a solução a 37 ° C num banho de água. </li><li> Lavam-se as células com PBS pré-aquecido para remover as esterases do soro. Aspirar o PBS. </li><li> Aplicar a solução pré-aquecida de trabalho sobre as células durante 2 minutos e lavá-lo com PBS. </li><li> Uso de microscópio de epifluorescência a imagem das células (PI ex λ = 490 nm, em λ = 570 nm; FDA ex λ = 490 nm, em λ = 514 nm). A mancha permanece estável ao longo de 40 min. Coloração prolongado pode resultar em coloração inespecífica resultante da absorção pelas células PI e de co-localização de PI / FDA em células. </li></ol></li><li> Immunostaining <ol><li> No ponto de tempo de cultura desejado fixar as células com paraformaldeído a 4% (PFA) durante 30 min à TA. Devido à toxicidade do PFA emanações o passo de fixação deve ser realizada na hotte química. </li><li> Lavar 3x com PBS andaimes Ponto de paragem:. As construções de PFA-fixadas podem ser armazenadas em PBS a 4 ° C durante vários dias antes de prosseguir com outros passos. </li><li> Incubar os andaimes em 0,2% de Triton, 0,25% de albumina de soro bovino (BSA) em PBS contendo anticorpo primário O / N a 4 ° C. </li><li> No dia seguinte descartar a solução de coloração e lavar os andaimes 3 x 30 min com PBS para remover o anticorpo primário não ligado. </li><li> Incubar as amostras com anticorpo secundário diluído em 0,2% de Triton, 0,25% de BSA em PBS durante 2h à TA. </li><li> Remover a solução de coloração e lavar os andaimes 3 x 30 min com PBS para remover o anticorpo secundário não ligado. </li><li> Imagem andaimes utilizando um microscópio confocal com objetivo 20X, tendo 100 mm z-seções da amostra com 1 um passo. Para visualizar as neurites finas a resolução da imagem deve ser de no mínimo 1.024 x 1.024 pixels. </li></ol></li><li> ARN, ADN e o isolamento das proteínas Nota: RNA, DNA e isolamento de proteínas é realizada utilizando um ADN comercial AllPrep / ARN / proteína Mini Kit de acordo com as instruções do fabricante. <ol><li> Utilizando uma pinça estéril transferir os andaimes de placa de cultura para um tubo de 2 ml estéril. Coloque um andaime por tubo. </li><li> Adicionar 200 ul de tampão RLT a cada tubo. </li><li> Disrupt a construção fragmentando-o com microtesoura estéreis. </li><li> Para homogeneizar o tecido perturbado, transferir o conteúdo do tubo para a coluna de centrifugação. Girar por 2 min a toda a velocidade numa micro.O lisado é homogeneizado à medida que passa através da coluna de spin. </li><li> Recolhe-se o fluxo através e utilizá-la imediatamente para isolar o ARN, ADN e proteína, seguindo o protocolo do fabricante. </li><li> Quantificar a produção dos ácidos nucleicos de qualquer outro método compatível (por exemplo, NanoDrop). </li><li> Quantificar a produção de proteína utilizando o BCA Protein Assay de acordo com as instruções do fabricante. Medir o resultado de ensaio usando leitor de microplacas a 562 nm de comprimento de onda. Nota: O rendimento esperado de ácidos nucleicos e proteínas, por construção, em relação à densidade de células é mostrada na Figura 4. </li></ol></li></ol>

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A publicação deste vídeo-artigo é patrocinado pela Corning.

Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

O sistema nervoso central (SNC) pode ser afectada por uma variedade de desordens que envolvem vascular, estrutural, funcional, infecciosas ou degenerativas. Estima-se que 6,8 milhões de pessoas morrem a cada ano em conseqüência de distúrbios neurológicos, o que representa um fardo socioeconômico crescente em todo o mundo 1. No entanto, apenas algumas das desordens têm tratamentos disponíveis. Portanto, há uma necessidade crítica de estratégias terapêuticas inovadoras para pacientes que sofrem de distúrbios neurológicos. Infelizmente, muitas terapias CNS segmentados falhar durante os ensaios clínicos, em parte devido à utilização de modelos de investigação pré-clínica inadequados, que não permitem a avaliação dos impactos agudos e crônicos com leituras funcionais fisiologicamente relevantes. Apesar dos esforços de investigação significativos nas últimas décadas, há uma vasta quantidade desconhecida sobre a estrutura e função do SNC. A fim de ganhar mais conhecimento, modelos animais são freqüentemente nósEd para modelar estados patológicos, tais como lesão cerebral traumática (TBI) ou demência, especialmente em estudos pré-clínicos. No entanto, os animais a partir de seres humanos diferem significativamente tanto na anatomia do sistema nervoso central, bem como em função, a expressão do gene e metabolismo 2-4. Por outro lado, in vitro, culturas 2D são o método mais comum para investigar a biologia celular e são rotineiramente utilizado para descoberta de drogas. No entanto, as culturas de células em 2D falta a complexidade e relevância fisiológica em comparação com cérebro humano 5-7. Enquanto não há substituto para o baixo custo e simplicidade de estudos de cultura de células em 2D ou a complexidade fornecida por modelos animais, a engenharia de tecidos 3D poderia gerar melhores modelos de pesquisa para fechar a lacuna que existe entre o 2D in vitro e in vivo abordagens. Engenharia de tecidos 3D fornece condições experimentais mais fisiologicamente relevantes obtidos por 3D interacções célula-célula e sinais extracelulares previstos pelos andaimes biomaterial. Despite a evidência significativa por trás do valor das culturas 3D, existem atualmente apenas alguns modelos de tecido do sistema nervoso central, tais como 3D-tronco derivadas de células culturas organï¿½de 8-10, neurospheroids 11 e disperso culturas hidrogel 12,13. Métodos de técnicas avançadas de multicamadas, incluindo a litografia 14, e impressão em 3D 15 foram utilizados para o estudo do pulmão, fígado, rim e tecido. No entanto, existe a falta de modelos 3D do SNC que permitam o crescimento neuronal compartimentada, como imitando a arquitetura cortical e biologia. Separado do crescimento de neurites a partir de corpos de células neuronais foi anteriormente demonstrada em culturas 2D utilizando microfabricação 16,17 permitindo que o estudo de rastreio tracto axónio, o influxo de cálcio, arquitetura de rede e actividades. Esta idéia nos inspirou a desenvolver um tecido neural polarizada 3D onde os corpos celulares e tratos axonais estão localizados em diferentes compartimentos imitando a arquitetura em camadas do cérebro 18 </sup&gt;. A nossa abordagem é baseada no uso de design único andaime de seda que pode acomodar até alta densidade de células em um volume confinado e permite excrescência axonal de rede densa em um gel de colagénio. Aqui demonstramos o procedimento completo conjunto de tecido cerebral semelhante, incluindo a fabricação de andaime e cultura neuronal.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

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.

Sólido de seda esponjas pré-cortada com a forma de rosca representam uma ideia original e simples para conseguir uma arquitectura semelhante a tecido neural compartimentada (Figura 2A). A estrutura altamente porosa do andaime com o tamanho de poro de cerca de 500 um resultado em área superficial excepcionalmente elevada permitindo a sementeira e o crescimento de elevada densidade celular dentro de um pequeno volume (2x10 7 / ml) (Figura 2B). Além disso, a elevada porosidade do andaime permite a difusão livre de nutrientes e resíduos que resultam em viabilidade superior de culturas celulares densas sobre quadros de tempo prolongado. Após a sementeira dos neurónios rápida e uniformemente aderem à superfície dos poros de andaime. Após a fixação das células está concluída e as células são equilibradas sobre o substrato de seda, o andaime esponja é preenchido com a matriz de colagénio macio, a fim de proporcionar um ambiente 3D para a formação da rede axonal (Figura 3 </strong&gt;). Na ausência de matriz de colagénio a compartimentalização dos axónios e corpos celulares não é possível, como neurónios extensões crescer apenas na superfície dos poros, resultando em redes mistas como encontrado com superfícies 2D (Figura 3B). Em contraste, o gel de colagénio proporciona uma malha que suporta grande crescimento axonal em 3D, enquanto que os corpos celulares neuronais permanecer ligado ao andaime de seda (Figura 3C). Este padrão de crescimento conduz a compartimentalização de corpos celulares axonais e redes puros (Figura 3D), o qual se assemelha a matéria cinzenta e branca do córtex do cérebro. Além de microscopia, as construções podem ser avaliadas com uma variedade de outros métodos de 18, dependendo da questão por trás do experimento. Por exemplo, o isolamento de ADN, ARN e proteína a partir das construções podem ser facilmente realizados utilizando kits comerciais (Figura 4). O rendimento de ácidos nucleicos e de proteínas PER construção depende do número de células inicialmente semeadas em andaimes de seda. Quanto mais células semeadas quanto maior o rendimento de ADN, ARN e proteína. <img alt="figura 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> Figura 1. PTFE molde utilizado para a preparação esponja seda andaime As dimensões:.. 10 cm de diâmetro, a 2 cm de altura <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> Figura 2. Modelo 3D tecido cerebral-like. (A) poroso esponja seda andaime. (B) Live / coloração de neurônios mortos no dia 1 após a semeadura na seda andaime antes de colágeno incorporação (células verdes-live, células red-mortos). Painéis sobre lado direito mostrar a magnification da área capturada em molduras vermelhas. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> Figura 3. Neuronal conseqüência compartimentada no modelo de tecido cerebral do tipo 3D (A) compartimentos: Andaime. Compartimento corpo de células Red, Blue-compartimento axonal. (B) padrão de crescimento neuronal no dia 1 após a semeadura antes incorporação de colágeno. (C). Crescimento neuronal no dia 7 após a sementeira no compartimento do corpo da célula. (D) excrescência Neuronal no dia 7 após a semeadura no compartimento axonal. Green -. ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta</a>figura. <img alt="Figura 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> Figura 4. Rendimento esperado de (A) RNA e DNA, e proteínas (B) por construção em relação à quantidade de células semeadas por andaime (± SD, n = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura .</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

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

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 ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Research

JoVE Journal

Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo elétrico controlado de migração dirigida de células progenitoras neurais em ambientes 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Bioengenharia

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Metodologia Elastografia por Ressonância Magnética para a Avaliação de engenharia de tecidos Crescimento Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocolo Relativo Avaliação hidrodinâmica de Tri-folheto polímero Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Geração e Alongamento das embarcações da engenharia de tecidos em um modelo de rato

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

A Tissue Combinada 3D Engineered<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Modelo de tumor de pulmão para prever a eficácia da droga em fundos específicos de mutação

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting da cartilagem e Analogs de tecido de pele utilizando um romance passiva unidade técnica de mistura para Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

A propagação e a implantação de um enxerto traqueal biossintética engenharia de tecido em um modelo do rato

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Enxerto projetado por tecido para reconstrução circunferencial do Esôfago em Ratos

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...