Os autores reconhecem o seguinte fonte de financiamento: o fundo magia que questões de investigação Cardiovascular.

1. preparação dos Cardiomyocytes
<ol><li>Placas de 6 boas casaco com membrana basal matriz e cultura induzida por humanos células estaminais pluripotentes (hiPSCs) como descrito anteriormente17.</li>
	<li>Diferencie hiPSCs em hiPSC-CMs usando métodos anteriormente descritos,18.</li>
	<li>Pós-diferenciação d 16-18, suspender a aplicação do cardiomyocytes enxaguando cada poço com 2 mL de tampão fosfato salino (PBS) de 1x sem cálcio ou magnésio, seguido de incubação com 1ml/bem de tripsina ou célula reagente de dissociação (ver tabela de de Materiais) por 5 min à temperatura ambiente.</li>
	<li>Neutralizar o reagente de dissociação de tripsina ou célula (veja a Tabela de materiais) usando um volume igual de mídia de célula de Roswell Park Memorial Institute (RPMI) suplementado com B-27 (mídia de células RPMI/B-27). Pipete para cima e para baixo para afrouxar as células aderidas.</li>
	<li>Recolher o cardiomyocytes suspenso com uma pipeta sorológica de 10 mL e transfira para um tubo cónico de 50 mL.</li>
	<li>Centrifugar a suspensão de eritrócitos a 250 x g durante 5 min à temperatura ambiente para obter um centrifugado.</li>
	<li>Resuspenda o pellet em 10 mL de RPMI/B-27 mídia de célula.</li>
	<li>Combinar a 20 µ l de suspensão de células com uma quantidade igual de solução de azul de Tripan 0,4% e misture delicadamente.</li>
	<li>Use um hemocytometer manual para contar e obter a concentração e a célula viabilidade da suspensão celular.</li>
</ol>2. preparação dos fibroblastos
<ol><li>Inicie uma cultura de uma linhagem de células humanas fibroblastos cardíacos (HCF) (tipo ventricular adulto) conforme descrito anteriormente,16.</li>
	<li>Suspender os HCFs incubando-os com uma quantidade adequada de tripsina ou célula reagente de dissociação (ver Tabela de materiais) durante 5 min à temperatura ambiente16. Para um balão de T175, utilizou-se 10 mL de reagente de dissociação de tripsina ou célula.</li>
	<li>Neutralizar o reagente de dissociação de tripsina ou célula (veja a Tabela de materiais) usando um volume igual de médio, então transferir a amostra para um tubo cónico de 50 mL e centrifugar a suspensão de eritrócitos a 250 x g durante 5 min à temperatura ambiente para obter uma pelota.</li>
	<li>Resuspenda o pellet em 10 mL de meio de crescimento do fibroblasto (ver Tabela de materiais).</li>
	<li>Combinar a 20 µ l de suspensão de células de HCF com uma quantidade igual de solução de azul de Tripan 0,4% e misture delicadamente.</li>
	<li>Use um contador de célula automatizada ou manual hemocytometer para contar e obter a concentração e célula viabilidade da nova suspensão celular.</li>
</ol>3. preparação de células endoteliais
<ol><li>Inicie uma cultura de uma linhagem de células endoteliais (HUVEC) de veia umbilical humana conforme descrito anteriormente,17. Suspender o HX incubando-os com uma quantidade adequada de tripsina ou célula reagente de dissociação (ver Tabela de materiais) para 3 min a temperatura da sala17. Neutralizar o reagente de dissociação de tripsina ou célula (veja a Tabela de materiais) usando um igual volume de meio, em seguida, transferir para um tubo cónico de 50 mL e centrifugar a suspensão de eritrócitos a 250 x g durante 5 min à temperatura ambiente para obter uma pelota. Nota: Para um balão de T175, 10 mL de reagente de dissociação de tripsina ou célula foi usado.</li>
	<li>Resuspenda o pellet em 10 mL de meio de cultura de células endoteliais (ver Tabela de materiais).</li>
	<li>Combinar a 20 µ l de suspensão de HUVEC e manchá-la com uma quantidade igual de solução de azul de Tripan 0,4% e misture delicadamente.</li>
	<li>Use um contador automatizado de células ou um hemocytometer manual para contar e obter a concentração e a célula viabilidade da suspensão celular.</li>
</ol>4. criação de esferoides gota de suspensão
<ol><li>Coloque o enforcamento gota dispositivo que contém 850 microporos (cada um com um diâmetro de 350 µm) em placas de 6 boas estéril.</li>
	<li>Isolar os três tipos de células, como descrito acima: hiPSC-CMs, HCFs e HX. Combiná-los em uma proporção de 70% de iPSC-CMs, 15% HCFs e HX de 15% em um tubo cónico de 50 mL com mídia de células RPMI/B-27 com uma concentração de 2.475.000 células por mL.</li>
	<li>Dispense a 4 mL de suspensão de célula (2.475.000 células / mL) a cada poço de penhora ultra baixo pendurado sistema da gota, (com um diâmetro de micropore de 350 µm), sentado em uma placa de 6.</li>
	<li>Esferoides formarão espontaneamente dentro de 12 h de dispensar a suspensão de células para o enforcamento dispositivo de gota. Continue a cultura para um total de 72 h (3-d), a 37 ° C, umidade de 95% e 5% CO2para a maturação dos esferoides.</li>
	<li>Após 72 h da cultura, colher os esferoides através dos poros na parte inferior do enforcamento descarta sistema colocando o dispositivo em um prato com médio e rodando-o suavemente para liberar os esferoides de enforcamento dispositivo de gota.</li>
</ol>5. 3-d Patch criação usando o molde líquido romance
<ol><li>A base, placa quadrada de fundo, fundo líquido e 10 lado em camadas, as redes são montadas para criar o romance molde com o auxílio de um par de Pinças esterilizadas. Primeiro, empilhar e alinhe a base, a placa de fundo quadrado e inferior a net usando os bornes de canto. Em seguida, pilha e as redes de 10 lado alternando as direções para criar uma rede de pinos de aço fino de camada.</li>
	<li>Irrigue o enchimento da base com PBS ou meio para reduzir a tensão superficial e, em seguida, transferir o molde montado sobre a base de enchimento.</li>
	<li>Molhe a cavidade do molde líquido com PBS ou médio no mesmo método. Encher lentamente os esferoides na cavidade pressuposta do tamanho desejado (2 x 2 x 1 mm, 4 x 4 x 1 mm, ou 6 x 6 x 1 mm). Nota: Certifique-se que 120% do volume previsto de esferoides é alimentado na cavidade, para que os agregados de células estão em conexão apertada e permanecer estáticos.</li>
	<li>Montar a rede top e top placa quadrada sobre os bornes de canto e fixe as rolhas e tubos de exploração para evitar um fiasco dos esferoides.</li>
	<li>Transferi o sistema de molde líquido enchido para uma placa de 6 com 6 mL de RPMI/B-27 mídia de célula ou em um recipiente estéril com suficiente médio para cobrir o molde todo montado.</li>
	<li>Incube a placa com o sistema de molde preenchido num agitador balançando 6 7-10 dias a 3.000-4.000 x g. Nota: A duração da incubação é decidida pelo tamanho dos patches cardíacos. Com manchas maiores, o tempo de incubação precisará ser aumentada para maturação de remendo cardíaca.</li>
</ol>6. remoção do Patch do romance molde líquido
<ol><li>Remover o sistema de molde da mídia e coloque-o sobre o tapete de manipulação esterilizado em um prato de 10 cm estéril.</li>
	<li>Remova o tubo de exploração, rolhas, a placa superior do quadrado e net top. Cuidadosamente retire as redes de lado em camadas, um a um com um par de Pinças esterilizadas. Após a descanulação, um remendo cardíaco intacto é obtido no topo do líquido do fundo.</li>
	<li>Buscar o fundo líquido com o patch usando pinça estéril e transferir o líquido do fundo em um prato de 35 mm com 5 mL de RPMI/B27 mídia. Afrouxe delicadamente o patch do fundo líquido agitando lentamente na net no prato, ou com um raspador de pilha de estéril. Após o desprendimento do fundo líquido, o tecido cardíaco pode ser cultivado com RPMI/B27 mídia.</li>
	<li>Continue a incubar o patch cardíaco 3D flutuante no prato 35mm. Batendo síncrono funcional pode ser observado logo em 24 h após a remoção do molde.</li>
	<li>Depois de remover o patch do molde líquido, observe que sua forma é mantida com a integridade e a intensidade de espancamento síncrona aumenta com o tempo. Nota: Os patches cardíacos baseada em molde líquidos 3D exibiram integração elétrica do componente cardiospheres após a descanulação. Observamos uma surra frequência variando de 60 a 80 batimentos por minuto que persistiu por mais de 60 dias.</li>
</ol>

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Os autores não têm nada para divulgar.

O significado deste método reside na sua reprodutibilidade e a eficácia do tecido cardíaco resultante em várias camadas. No campo da engenharia de tecido cardíaco, uma das metas do atuais é identificar um método para construir batendo, várias camadas e funcionais patches cardíacas em 3D. Nós relatamos um método eficiente e reprodutível de criação de várias camadas de tecidos cardíacos por sementeira directa manual de esferoides compostas de fibroblastos, células endoteliais e cardiomyocytes num romance m...

O objetivo da engenharia de tecido cardíaco atual é desenvolver uma terapia para substituir ou reparar a estrutura e função do tecido lesado do miocárdio1. Métodos para criar modelos 3D de tecido cardíaco, exibindo as propriedades contráteis e eletrofisiológicas importantes do tecido cardíaco nativo tem sido rápida expansão2,3. Uma variedade de estratégias foram explorados e utilizados em estudos de4,5. Esses métodos variam o uso do específico hidrogel bioativos naturais e sintéticas, tais como gelatina, colágeno, fibrina e peptídeos6, a bio-tinta deposição tecnologias2 e bioprinting tecnologias7.
Ficou demonstrado que métodos livre de andaime podem produzir tecidos comparáveis como métodos baseados em biomateriais, sem os inconvenientes de incorporação de material estrangeiro andaimes8. Oren Caspi et al demonstraram que a incorporação de vários tipos de células permite a geração de tecido cardíaco engenharia humano altamente vascularizado9. Chin et al desenvolveram um método de impressão 3D para criação de remendo cardíaca de esferoides. Resultante de patches são compostas de cardiomyocytes, fibroblastos e células endoteliais em uma relação de 70:15:1510. Esferoides foram mostrados para ser eficazes "blocos de construção" da criação de tecido cardíaco andaime-livre, como eles são resistentes contra hipóxia e possuem integridade mecânica suficiente para implantação de11,12. Estudos anteriores têm demonstrado vários métodos de fabricação para criação de esferoide, incluindo o uso do enforcamento soltar método, girador frascos13, microfluidic sistemas14e superfícies não-aderente cultura não revestidos ou revestidos com micromoldes de agarose15. Neste protocolo, usamos o enforcamento gota dispositivo, que contém microporos para centenas de esferoides de condensação de uma só vez.
Este estudo apresenta uma nova e eficiente método de andaime-livre para a criação de tecido cardíaco, que inclui a semeadura manualmente os esferoides em uma cavidade do molde quadrado e incubando o tecido num agitador para maturação. Em condições habituais de cultura estática, difusão de oxigênio é limitada aos aspectos exteriores da construção de tecido, resultando em necrose central. No entanto, com o molde líquido, todos os esferoides semeados no molde estão imersos na mídia com um constante movimento fluídico, permitindo a maior difusão de nutrientes e oxigênio. Além disso, este método baseado em molde permite a criação simultânea de patches de tecido de diferentes tamanhos, com o mínimo de esforço manual e o tecido resultante pode ser facilmente removido do molde. Este novo método permite a criação eficiente e reprodutível de patches cardíacas andaime-livre, em várias camadas.

<table border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Human Cardiac fibroblasts (HCF)</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">6310</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">FM-2 Consists of Basal Medium</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">2331</td>
			<td style="width:220px;">HCF culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Human umbilical vein endothelial cells (HUVEC)</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EGM+Bullet Kit&#160;</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC5035</td>
			<td>HUVEC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">E8 media&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1517001</td>
			<td>HiPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Geltrex&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TrypLE Express Enzyme (1X)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">12604013</td>
			<td>Trypsin and Cell dissociation reagent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">RPMI media</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11875093</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">B-27 supplement (50x)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">17504044</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Novel net mold&#160;</td>
			<td style="width:139px;">TissueByNet Co.,Ltd</td>
			<td style="width:119px;">NM25-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hanging drop plate</td>
			<td style="width:139px;">Kuraray Co.,Ltd</td>
			<td style="width:119px;">MPc350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6 well plates&#160;</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS-3516</td>
			<td></td>
		</tr>
	</tbody>
</table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

Este protocolo descreve um romance e método baseado no molde líquido fácil criar tridimensional (3D) cardíaca os tecidos sem material adicional do andaime. Cardiomyocytes de tronco-derivado de células pluripotentes induzidas pelo homem (iPSC-CMs), fibroblastos cardíacos humanos (HCFs) e células endoteliais da veia umbilical humana (HX) são isoladas e usadas para gerar uma suspensão de células com 70% iPSC-CMs, HCFs 15% e 15% HX. Eles são co cultivados em um sistema de fixação ultra baixo "gota de suspensão" que contém microporos para centenas de esferoides de condensação de uma só vez. As células agregam e formam espontaneamente esferoides surra após 3 dias de co-cultura. Os esferoides são colhidas, semeados em uma cavidade do molde de romance e cultivadas num agitador na incubadora. Os esferoides tornar-se um tecido maduro funcional, aproximadamente 7 dias após a semeadura. Os tecidos resultantes de várias camados consistem de esferoides fundidos com integridade estrutural satisfatória e comportamento batendo síncrono. Este novo método tem um potencial promissor como um método reprodutível e de baixo custo para criar tecidos projetados para o tratamento da insuficiência cardíaca no futuro.

Em nossos experimentos, utilizamos uma suspensão de células de iPSC-CMs de 70% e 15% HCFs HX de 15% na mídia de células RPMI/B-27 com uma concentração de 2.475.000 células por mL. Depois de criar a suspensão de eritrócitos, demitiremos 4 mL de suspensão de célula para cada poço de penhora ultra baixo pendurado o sistema de entrega, conforme descrito na etapa 4.3 do protocolo. O uso do enforcamento gota sistema resultou na formação espontânea de centenas de bater esferoides ...

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

Um método baseado em molde líquido de criação livre de andaime tridimensional de tecido cardíaco

Este protocolo descreve um método baseado em molde líquido para criar tecidos cardíacos livre de andaime tridimensionais com integridade estrutural satisfatória e comportamento batendo síncrono.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

Research

JoVE Journal

Bioengineering

8.8K visualizações.  The First Hospital of Jilin University. Este protocolo descreve um método baseado em molde líquido para criar tecidos cardíacos livre de andaime tridimensionais com integridade estrutural satisfatória e comportamento batendo síncrono.

Vídeo: A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional Optical resolução Microscopia Fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bioengenharia

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impressão de planar e tridimensional de tintas condutoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Cartilagem Human Tissue Fabricação Usando tridimensional Inkjet Tecnologia de Impressão

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabricação de Inverted coloidal de cristal poli (etileno-glicol) Andaime: Uma Plataforma Cultura celular tridimensional para Liver Engenharia de Tecidos

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Criação de tecido cardíaco exibindo integração mecânica de esferóides usando 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Engenharia de Tecido Tridimensional de Redes de Astrócitos Alinhadas para Recapitular Mecanismos de Desenvolvimento e Facilitar a Regeneração do Sistema Nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamíferos em andaimes tridimensionais do Peptide

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Microdissection de segmentos de tecido Renal primária e incorporação com tecnologia de construção romance livre de andaime

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...