Agradecemos T. Hunt, M. Rosenbluth, eo Laboratório de Lam para os seus conselhos e discussões úteis. Agradecemos o apoio de G. Spinner e do Instituto de Eletrônica e Nanotecnologia do Georgia Institute of Technology. O apoio financeiro para este trabalho foi fornecida por uma subvenção NIH K08-HL093360, prêmio UCSF REAC, um Nanomedicina NIH Desenvolvimento Award Centro PN2EY018244 e financiamento do Centro de Biologia Celular endotelial de Saúde Infantil de Atlanta.

1. Fabricação de microdispositivos o endotelial <ol><li> Criar um fotomáscara enviando um desenho assistido por computador (CAD) do dispositivo micro a um fornecedor máscara fora. A máscara utilizada foi composta de uma camada de cromo em refrigerante de vidro de cal. Neste caso, a largura do canal microfluídico foi de 30 uM. </li><li> Limpar uma bolacha de silício nu com piranha (10:1 proporção de ácido sulfúrico e peróxido de hidrogénio) durante 15 minutos e mergulhar em ácido fluorídrico por 30 segundos. Lavar com água desionizada (DI) durante aproximadamente 10 segundos. </li><li> Utilizando um revestidor de rotação, de spin Microchem SU-8 2025 fotorresiste sobre a bolacha de uma altura de 30 mm. Para SU-8 2025, uma velocidade de centrifugação de 3000 rpm é recomendada. Outros viscosidades de SU-8 estão disponíveis, que irá atingir esta altura. As instruções completas para SU-8 uso estão disponíveis em <a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> Coloque a bolacha com SU-8 sobre uma placa de aquecimento a 95 ° C durante5 minutos para expulsar o excesso de solvente. Nota: Um forno irá secar a bolacha de modo diferente do que uma placa de aquecimento e não é recomendada. </li><li> Colocar uma máscara com a forma característica desejada sobre a bolacha, e expor à luz UV (160 mJ / cm 2 medido a 365 nm) em um alinhador de máscara (Karl Suss, MA-6). Esta cruz liga o fotorresiste. </li><li> Coloque a bolacha de volta numa placa de aquecimento a 95 ° C durante um período adicional de 5 minutos para acelerar ainda mais a polimerização de SU-8. </li><li> Mergulha-se o bolacha em SU-8 Developer, composto principalmente por PGMEA (propileno glicol metil éter acetato) durante 4 minutos para remover o não-reticulado SU-8. </li><li> Lavar a bolacha recentemente desenvolvido com 100% de álcool isopropílico (IPA) durante 10 s. A bolacha pode então ser seca usando azoto pressurizado ou permitindo a evaporação do solvente da bolacha em uma capa limpa fumos durante vários minutos. </li><li> Fita as bordas da bolacha seca com modelado SU-8 em um prato de petri de prédesabafar movimento. </li><li> Aplicar 1 mL de Sigmacote para a bolacha utilizando uma pipeta, a tampa com o topo da placa de petri, e bolacha redemoinho para assegurar um revestimento completo da bolacha, remover cobrir, e permitir que bolacha para secar durante vários minutos até todo o solvente se ter evaporado. </li></ol> 2. PDMS Preparação (polidimetilsiloxano) <ol><li> Mistura PDMS polímero e agente de cura numa proporção 10:1 (w / w) e remover as bolhas de ar, utilizando um exsicador de vácuo. O período de tempo necessário para desgaseificar a PDMS varia com a força do sistema de vácuo disponível, mas tipicamente varia desde alguns minutos a uma hora. Para uma placa de Petri seis polegadas com nenhuma PDMS anteriormente vertida, num volume total de 60 mL de polímero é recomendada. </li><li> Despeje a mistura sobre a bolacha, a cerca de 5 mm de espessura. Deita-se também um prato de fundo plano para criar uma folha fina de PDMS, cerca de 1 mm de espessura. Curar a 60 ° C num forno durante a noite. </li><li> Usando uma faca ou bisturi, cortar ao redor do cured dispositivo PDMS e removê-lo a partir da bolacha. Além disso, cortado de uma folha fina de PDMS ligeiramente maior do que o dispositivo. </li><li> Criar entrada e de saída furos no dispositivo PDMS utilizando um furador de 1,0 mm. Isto pode ser conseguido usando um vice-um pino, o dispositivo de Harris Uni-Core, ou similar. </li><li> Limpar as superfícies do dispositivo ea folha usando fita adesiva. </li><li> Usando um limpador de plasma, expor as superfícies do dispositivo PDMS e folha de PDMS ao plasma de oxigénio durante 30 s. O plasma de oxigénio cria espécies reactivas na superfície exposta do PDMS que se ligam quando postos em contacto físico. </li><li> Ligue menor tubagem com um comprimento pequeno (vários centímetros) de tubo de grande, o que por sua vez está ligado a uma seringa com uma agulha romba de ponto, preenchido com 50 fibronectina ug / ml de plasma humano em PBS. A tubagem e da agulha são dimensionados de tal modo que um encaixe de fricção é criado entre o tubo. Insira menor a tubagem para a entrada do PDMS microdispositivo e umplicar pressão para a seringa para encher completamente os canais com uma solução de fibronectina e para criar uma queda de 100 uL pequena na porta de saída, para assegurar que os canais de ficar molhado. Incubar o dispositivo a 37 ° C durante 40-60 min. </li><li> Conecte uma seringa cheia de PBS fresca para a agulha de ponto cego. Aplicar pressão para a seringa para enxaguar o dispositivo com PBS. </li></ol> 3. Semeando a dispositivos microfluídicos com células endoteliais <ol><li> Preparar 1.000.000 células / ml de células endoteliais da veia umbilical humana (HUVECs) em meios de crescimento endotelial com dextrano 8%. Uma gama de 500.000 a 2.000.000 células / ml foi utilizado com sucesso. A adição de dextrano ao meio de carga célula aumenta a viscosidade do fluido, o que diminui a velocidade de células endoteliais como eles entram no sistema fibronectina revestido microfluídicos. Isto, por sua vez, aumenta a probabilidade de que as células irá aderir e cultura com sucesso dentro do microdispositivo. </li><li> CONECTE a nova seringa e tubagem como no Passo 2,7. mas com um tempo mais tubagem (aproximadamente 1 metro de comprimento). </li><li> Para optimizar o desempenho do presente sistema, é crítica neste ponto para evitar qualquer fuga ou bolhas em todo o sistema de perfusão; qualquer fuga de solução ou a presença de bolhas nos meios de comunicação irá alterar o fluxo e evitar a semeadura bem sucedida de células endoteliais. Portanto, apertadas do tubo é essencial para eliminar vazamentos entre as conexões. Bolhas na seringa e / ou da tubagem devem ser eliminadas antes células são introduzidas no microdispositivo e todo o sistema de perfusão devem ser preparadas com a solução de células antes de prender o tubo à microfluídico para evitar a introdução de bolhas de ar. </li><li> Utilizando uma bomba de seringa, infundir suspensão de células em PDMS dispositivo a uma taxa de fluxo volumétrica de 1,23 ul / min durante 2 horas a 37 ° C e 5% de CO 2. Óptimos resultados foram obtidos quando a bomba de seringa estava no<html> mesma altura que o dispositivo de PDMS. Para a entrada, o mais tubos, que pode ser enrolada dentro da incubadora, assegura que as células e meios de comunicação estão adequadamente aquecido antes de entrar em contacto com o dispositivo. No nosso sistema, a taxa de fluxo volumétrica de 1,23 ul / min aproxima uma velocidade midstream de ~ 1 mm / s nos menores microcanais, correspondendo a uma tensão de cisalhamento de parede de ~ 1 dine / cm 2 a esses canais utilizando sangue total. </li><li> Usando a bomba de seringa mesmo e tubagem de comprimento, perfundir meios de crescimento fresco para 2-8 dias à razão de 1,23 ul / min. Um dispositivo de sucesso terá uma monocamada de células endoteliais que crescem no interior do dispositivo dentro de 24-48 horas. As experiências anteriores demonstram que monocamadas confluentes apropriadamente expressar VE caderina-a célula-célula junções em todo o dispositivo 14. </li><li> Injectar sangue ou suspensão de células no sistema para a experimentação. </li></ol> 4. Os resultados representativos _content &quot;&gt; Usando este protocolo, técnicas padrão de microfabricação litográficas são usados ​​para criar o molde necessária para produzir os canais microfluídicos que imitam a fisiologicamente sizescale da microvasculatura (Figura 1A). Usando uma técnica de perfusão optimizado, as células endoteliais, em seguida, a cultura de sementes e confluently o superfície interna inteira do sistema microfluídico dentro de 24-48 horas de semeadura de células (Figura 1B). Como o sistema microfluídico é transparente, o microdispositivo inteiro pode ser colocado sobre um campo claro / etapa microscópio de fluorescência para imagiologia e recolha de dados. O nosso sistema pode então ser aplicado para estudar doenças hematológicas que envolvem alteradas as propriedades biofísicas, tais como a doença de células falciformes, em que o aumento da rigidez falciformes células vermelhas e adesão de leucócitos e endotelial aberrante contribuir para a obstrução microvascular. Um medicamento aprovado clinicamente, hidroxiuréia, melhora sintomas, mas a sua direct efeito sobre o fluxo microvascular é desconhecida. Nosso ensaio leva em conta tanto a rigidez e adesão celular, e demonstra que hidroxiureia significativamente melhora o fluxo na doença de células falciformes (Figura 2). A doença falciforme é apenas um exemplo de uma aplicação para a microvasculatura-on-a-chip, como este sistema é ideal para estudar qualquer processo hematológica em que as células do sangue interagem uns com os outros e as células endoteliais da microvasculatura. Outras aplicações clinicamente relevantes incluem distúrbios inflamatórios, sepse / lesão pulmonar, microangiopatias trombóticas, malária e metástase de câncer, enquanto aplicações mais básicas incluem a biologia de leucócitos e da biologia de células estaminais hematopoiéticas, entre muitos outros. <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="A Figura 1"/> Figura 1. A) o PDMS inicial dispositivo micro antes endotelização. Aqui, o microdispositivo é injetado a sagacidadeh corante alimentar para ilustrar desenho sizescale e global do sistema. B) microscopia de campo claro mostra o sistema de microfluídico é completamente endothelialized dentro de 48 horas de semeadura célula utilizando o protocolo aqui descrito. <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="A Figura 2"/> Figura 2. A) A microvasculatura-em-um-chip com microcanais aproxima do tamanho de vénulas pós-capilares (30 mm), o local da maioria das células falciformes eventos obstrutivos microvasculares. Sangue total de pacientes com anemia falciforme que receberam hidroxiuréia e de pacientes que não receberam hidroxiuréia flui através de dois diferentes microvasculatura-on-a-chip dispositivos. B) O sangue total de pacientes com anemia falciforme que recebem fluxos de hidroxiureia com relativa facilidade dentro dos microcanais endothelialized. C) Sob as mesmas condições hemodinâmicas, o sangue total a partir de pacientes com anemia falciforme que não recebem hidroxiureia, no entanto, apresenta fluxo muito mais lento com microchobstrução Annel. O canal de fundo é completamente obstruído com ausência de fluxo e as velocidades do fluxo no microcanais outros endothelialized são significativamente mais baixos do que na condição de hidroxiureia. A barra de escala em todas as três imagens é 30 mm.

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O nosso sistema microdispositivo endothelialized é mais adequada quando usado em conjunção com as experiências in vivo, ea sua abordagem reducionista pode ajudar a elucidar os mecanismos biofísicos de processos hematológicas que são observadas em humanos e em modelos animais. Além disso, nosso sistema não é sem limitações. Por exemplo, os nossos canais microfluídicos são quadrados em corte transversal. Embora tecnicamente microcanais circulares podem ser fabricados 10,11, optou...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> agulha ponto sem corte </td><td> OK Internacional </td><td> 920050-TE </td><td> Precision TE agulha calibre 20 x 1/2 &quot;, cor de rosa </td></tr><tr><td> dextrano </td><td> Sigma-Aldrich </td><td> 31392 </td><td></td></tr><tr><td> A fibronectina </td><td> Sigma-Aldrich </td><td> F0895 </td><td></td></tr><tr><td> Furador (morsa pinos) </td><td> Inovações técnicas </td><td></td><td></td></tr><tr><td> As células endoteliais do cordão umbilical humana (HUVECs) </td><td> Lonza </td><td> CC-2519 </td><td></td></tr><tr><td> Limpador de Plasma </td><td> Plasma </td><td> PDC-326 </td><td></td></tr><tr><td>Polidimetilsiloxano (PDMS) </td><td> Fisher Scientific </td><td> NC9285739 </td><td> Sylgard 184 KIT Elastômero de Silicone </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2 </td><td></td></tr><tr><td> SU-8 2025 </td><td> Microchem </td><td> Y111069 </td><td></td></tr><tr><td> SU-8 Desenvolvedor </td><td> Microchem </td><td> Y020100 </td><td></td></tr><tr><td> Bomba de seringa </td><td> Harvard Apparatus </td><td> 70-3008 </td><td> PHD-ULTRA </td></tr><tr><td> tubagem (maior) </td><td> Cole-Parmer Instrument Company </td><td> 06418-02 </td><td> Tygonreg tubulação microboro, 0,020 &quot;x 0,060 ID&quot; OD </td></tr><tr><td> tubagem (menor) </td><td> Cole-Parmer Instrument Company </td><td> 06417-11 </td><td> PTFE tubulação microboro, 0,012 &quot;x 0,030 ID&quot; OD </td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

Os avanços nas técnicas de microfabricação permitiram a produção de baixo custo e reprodutível sistemas microfluídicos para a realização de experiências biológicas e bioquímicas no 1,2 micro e nanoescala. Além disso, microfluídica também têm sido usados ​​especificamente para analisar quantitativamente processos hematológicas e microvascular, devido à sua capacidade para controlar facilmente o ambiente dinâmico fluídica e condições biológicas 3-6. Como tal, os investigadores têm utilizado sistemas mais recentemente microfluídicos para estudar deformabilidade de células do sangue, a agregação de células sanguíneas, o fluxo sanguíneo microvascular, e sangue interacções célula-célula endotelial 6-13. No entanto, estes sistemas microfluídicos quer não incluem células endoteliais cultivadas ou eram maiores do que a correspondente sizescale para microvasculares processos patológicos. Uma plataforma microfluídica com cultura de células endoteliais que recapitula com precisão o celular, físico e hemodynambiente âmico da microcirculação é necessário para promover nossa compreensão da fisiopatologia subjacente biofísico de doenças hematológicas que envolvem a microvasculatura. Aqui, apresentamos um método para criar uma &quot;endothelialized&quot; modelo in vitro da microvasculatura, usando um processo simples de microfabricação única máscara em conjunto com as técnicas de cultura de células endoteliais, estudar as interações patológicas microvasculares biofísicos que ocorrem na doença hematológica. Este &quot;microvasculatura-on-a-chip&quot;, prevê o pesquisador, com um teor robusto que controla rigidamente biológica, bem como condições biofísicas e é operado através de uma bomba de seringa padrão e de campo claro / microscopia de fluorescência. Parâmetros como condições hemodinâmicas da microcirculação, tipo de célula endotelial, o tipo de células sanguíneas (s) e concentração (s), drogas / concentração inibitória etc, tudo pode ser facilmente controlada. Como tal, a nossa microsistema forneceum método para investigar quantitativamente processos de doença no qual o fluxo microvascular é prejudicada devido a alterações na adesão celular, agregação e deformação, uma capacidade disponível com os ensaios existentes.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Um método para a cultura de uma monocamada de células endoteliais em toda a superfície interior 3D inteiro de um dispositivo micro com microvascular de tamanho canais (= 30 mm) é descrito. Este<em&gt; In vitro</em&gt; Modelo microvasculatura permite o estudo das interacções entre as células biofísicas do sangue, células endoteliais, e factores solúveis em doenças hematológicas.

A microfluídica Endothelialized para estudo das interacções microvasculares em Doenças Hematológicas

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

Research

JoVE Journal

Bioengineering

15.8K visualizações.  Emory University School of Medicine. Um método para a cultura de uma monocamada de células endoteliais em toda a superfície interior 3D inteiro de um dispositivo micro com microvascular de tamanho canais (= 30 mm) é descrito. Este In vitro Modelo microvasculatura permite o estudo das interacções entre as células biofísicas do sangue, células endoteliais, e factores solúveis em doenças hematológicas.

Vídeo: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Misturadores microfluídicos para Estudar o dobramento de proteínas

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Bioengenharia

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinação da taxa de transporte de xenobióticos e nanomateriais através da placenta usando o<em&gt; Ex vivo</em&gt; Modelo de perfusão da placenta humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Procedimento para o Desenvolvimento da Multi-profundidade Circular transversal reendotelizados microcanais-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

O uso de chips microfluídicos para Imagens ao vivo e estudo das respostas Lesão em<em&gt; Drosophila</em&gt; Larvas

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

A impressão Submerso de células em uma superfície modificada Usando um contínuo fluxo Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolor detecção de fluorescência para Gota microfluídica Utilizando Fibras Ópticas

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

Isolamento de Murino adiposo derivada de tecido microvascular Fragmentos como Vascularização Unidades para engenharia de tecidos

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microfluídica interfaceamento com matrizes de microeletrodos para estudar comunicação Neuronal e propagação de sinal Axonal

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Organóides neurais humanos para estudar câncer cerebral e doenças neurodegenerativas

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...