Este trabalho foi apoiado por uma concessão da pesquisa do California Institute for Quantitative Biosciences (QB3), o Bridging the Award Gap da fundação da família de Rogers, a Sandler / Programa Foundation UCSF para Breakthrough de Pesquisa Biomédica, uma bolsa da BASF, ea NSF, através Programa Faculdade Início de Carreira Desenvolvimento (CARREIRA) (DBI-1253293).

1. SU8 Mestre Fabrication <ol><li> Projetar as estruturas microfluídicos para dois fabricação camada utilizando o software AutoCAD e têm os desenhos impressos por um fornecedor em filme placa de circuito com resolução de 10 m. Os detalhes da concepção do dispositivo são dadas em uma referência anexado 11 e as geometrias de canal são mostradas na Figura 1. As camadas deverá incluir marcas de alinhamento para ajudar a colocar características de cada camada 12 de fabricação. </li><li> Coloque uma pré-limpeza de 3 polegadas de diâmetro wafer de silício em um revestidor de rotação e ligue o vácuo para afixá-lo para o mandril. Aplicar 1 ml de SU8-3035 no centro da bolacha e centrifugação durante 20 segundos a 500 rpm, em seguida 30 segundos a 2000 rpm, proporcionando uma espessura de camada de 50 um. </li><li> Remover a bolacha e assar em uma placa de aquecimento de 135 ° C durante 30 min. Permitir que o wafer arrefecer à RT antes de passar para a próxima etapa. </li><li> Exponha o wafer revestido para o st máscara de camada 1 (Figura 2A </strong&gt;) sob uma colimado 190 mW, 365 nm LED para 90 seg. Após a exposição, a colocar sobre uma bolacha de 135 ° C placa de aquecimento durante 1 min, em seguida, arrefecer attemperatura ambiente, antes de prosseguir para o passo seguinte. </li><li> Coloque o wafer no revestidor de rotação e ligue o vácuo para afixá-lo para o mandril. Aplicar 1 ml de SU8-2050 no centro da bolacha e centrifugação durante 20 segundos a 500 rpm, em seguida 30 segundos a 1375 rpm, o que resulta em uma camada que proporciona uma espessura adicional de 135 uM. </li><li> Remover a bolacha e assar em uma placa de aquecimento de 135 ° C durante 30 min, em seguida, arrefecer attemperatura ambiente, antes de passar para a próxima etapa. </li><li> Alinhar a camada de máscara 2 ND (Figura 2B) para a geometria modelada em 1.3 e expor a pastilha revestida a uma colimado 190 mW, a 365 nm LED durante 3 min. Após a exposição, colocar sobre uma placa de aquecimento de 135 ° C durante 1 min, em seguida, arrefecer attemperatura ambiente, antes de prosseguir para o passo seguinte. </li><li> Desenvolver as máscaras por imersão num banho em agitação de acetato de éter propilenoglicol monometílico, durante 30 min. Lava-se a bolachaem isopropanol e assar em uma placa de aquecimento de 135 ° C durante 1 min. Colocar o mestre desenvolvido numa placa de Petri de 100 mm para a moldagem por PDMS. </li></ol> 2. PDMS fabricação de dispositivos <ol><li> Preparar 10: 1 PDMS combinando 50 g de base de silicone com 5 g de agente de cura em um copo de plástico. Misture o conteúdo com uma ferramenta rotativa equipada com um pedaço de pau mexa. Desgaseificar a mistura no interior de um exsicador durante 30 minutos, ou até que todas as bolhas de ar são removidas. </li><li> Despeje o PDMS para dar uma espessura de 3 mm sobre o mestre e coloque de volta no secador durante mais de desgaseificação. Uma vez que todas as bolhas são removidas, o dispositivo de cozer a 60 ° C durante 2 horas. </li><li> Corte o dispositivo a partir do molde utilizando um bisturi e coloque sobre uma superfície limpa, com o lado modelado up. Cortar o molde PDMS ao meio com uma lâmina de barbear para separar master 1 da Master 2 (Figura 3a). Na peça que contém a geometria manuseio 50 um fluido impresso por Master 1, socos fluídicos as entradas e saídas com 0,75 mm de punção para biópsia. </li><li> Plasma tratar os dispositivos a 1 mbar de O2 no plasma durante 60 segundos em um aspirador de plasma 300 W. Molhar a superfície da peça unpunched de PDMS com uma gota de água Dl para retardar temporariamente PDMS-PDMS ligação e servir como um lubrificante. Durante a visualização através de um microscópio estéreo, lugar Master 1 no Master 2 superfície e deslize as superfícies relativamente até que um bloqueio mecânico é alcançada quando os quadros recesso e molduras salientes na Figura 3A companheiro. </li><li> Coloque o dispositivo num forno de 60 ° C e assar o dispositivo montado (Figura 3B) durante dois dias a 60 ° C para evaporar a água e a ligação completa. </li></ol> 3. Preparação dos Reagentes <ol><li> Encha seringa de 1 mL com água destilada para a fase interior. </li><li> Preencha seringa de 1 ml com HFE 7500 óleo fluorado com um peso. % Surfactante biocompatível surfactante 13 para a fase intermediária. </li><li> Preencha seringa de 10 ml com 10 wt. % Polietilenoe glicol (PEG) em solução de água contendo 1 em peso. % De Tween 20 e 1 em peso. % De dodecilsulfato de sódio para a fase contínua. </li></ol> 4. Sistema de Preparação <ol><li> Coloque o chip microfluídico no palco de um microscópio invertido acoplado com uma câmera digital capaz de &lt;100 ms velocidades do obturador. </li><li> Montar todas as seringas em bombas de seringa e anexar 27 agulhas G. Anexar ~ 30 cm de comprimento de tubagem de PE-2 sobre as agulhas e inserir as extremidades soltas na apropriado perfurados orifícios no dispositivo. </li><li> Inserir um comprimento de 10 cm de EP-2 para a porta de saída do dispositivo e colocar a outra extremidade num contentor de recolha de resíduos. </li><li> Primeiro o dispositivo, executando as bombas de seringa, a altas taxas de velocidades (2,000 mL / min) até que o fluido nos segmentos de tubagem atinge os orifícios de entrada do dispositivo. </li></ol> 5. Emulsão Geração <ol><li> Concentre o microscópio sobre uma região que contém o 50 um x 50 um orifício eocanal de saída a jusante. </li><li> Definir as bombas de seringa para fornecer o fluido para o gerador de emulsão dupla a taxas de fluxo de 250 mL / h durante a fase interior, com 100 ul / h durante a fase de meio, e 700 ul / h durante a fase contínua e esperar 10 minutos para equilíbrio. </li><li> Manter as velocidades de fluxo das fases interna e meia em 250? L / h e 100 l / h, respectivamente. Definir a taxa de fluxo da fase exterior em 1.050 mL / hr. Espere 3-5 minutos para a geração de casal emulsões para estabilizar sob este conjunto de condições de escoamento. </li><li> Adquirir 5 segundos de imagens de vídeo a 30 Hz para processamento off-line através de análise de imagem manual. </li><li> Repetir 5.3 e 5.4 com as taxas de fluxo indicadas na Tabela 1. As taxas de fluxo da fase interna e do meio são mantidas constantes e a taxa de fluxo da fase portadora é variada por ajustamento da configuração da bomba de seringa. </li></ol>

<ol>
	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. 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The authors have nothing to disclose.

A geometria de geração de emulsão dupla descrito aqui é projetado para imitar a física de dispositivos de capilares de vidro 8. Nestes, capilares de vidro cilíndricos alinhados são usadas para criar um jacto coaxial de três fases, que é cortado em gotículas uniformes de emulsão dupla. A função do nosso dispositivo de PDMS 3D é dependente do alinhamento central das características pequenas formadas com 50 mm de altura de fabricação da fase da portadora com canais que são de 320 mícrons na al...

<html> Emulsões duplas consistem em gotículas separadas a partir de uma fase de transportador por intermédio de uma camada de fluido, imiscível, e são de particular interesse devido a suas potenciais utilizações em aplicações industriais, produtos farmacêuticos e aplicações biológicas 1. Em alguns casos, a capacidade de encapsular compostos de alto valor em um núcleo de emulsão dupla permite que o material a ser protegido e lançado de uma maneira controlada. Por exemplo, as drogas podem ser encapsulados sob condições de solubilidade não apropriados para o fluido de transporte externo 2. Além disso, a camada intermédia do óleo pode ser usado como um modelo para a cápsula de encapsulamento e de entrega de drogas, cosméticos, e nutrientes 3. Em biologia, emulsões duplos também são úteis para triagem de alto rendimento, porque eles permitem um grande número de experiências sub-nanolitros a ser levada a cabo, e, em seguida, detectados utilizando um classificados de células activadas por fluorescência (FACS) instrumento 4,5. ENT &quot;&gt; O design de emulsões duplas com características de desempenho desejadas exige o controlo preciso do tamanho de emulsão dupla, composição e uniformidade. Embora os processos de emulsificação a granel, tais como emulsificação da membrana, são usadas na indústria, as emulsões resultantes são altamente polidispersos, exibindo um grande variedade de propriedades funcionais 1. O campo de gota de microfluídica é naturalmente adequadas a geração de emulsões monodispersas com composição cuidadosamente controlada 6. geração de emulsão dupla Microfluidic foi conseguido com duas estratégias principais, para preparar queda seqüencial e de fluxo capilar de vidro de focagem. emulsões duplas podem ser gerado em planares dispositivos PDMS usando uma gota de duas fases processo de tomada. Em primeiro lugar, aquosa-em-óleo são criados a partir de água-em-óleo região de um dispositivo com as paredes do canal hidrofóbico de tomada de gota. Em seguida, a emulsão pode ser fluiu ou reinjetado em uma região de tomada de gota com paredes hidrofílicos adequados para óleo em águagota de tomada 4. No entanto, o tratamento de superfície hidrófila de PMDS requer um passo de fabrico adicional e é muitas vezes temporária 7. O método mais controlável e reproduzível para formar emulsões duplas é por fluxo de co-axial de focagem, utilizando uma técnica pioneira Microfluidics capilar de vidro, pelo qual um jacto concêntrico contendo as três fases é cortado por meio de um pequeno orifício para produzir gotas monodispersas 8. Esta técnica permite a produção de gotículas muito mais pequenas do que as dimensões do canal, com o tamanho e a composição exacta da emulsão dupla é uma função das taxas de fluxo de cada fase. A grande diferença entre gota e tamanho do canal e do fluxo de bainha protetora externa impede gotas de entrar em contato com as paredes do canal, tornando desnecessário o tratamento de superfície. No entanto, esses dispositivos de vidro exigem fabricação personalizada de dicas de capilares afilados, juntamente com a montagem cuidadosa e selagem. Investigadores anteriores usaram litografia suave 3Dgrafia de gerar emulsões duplas utilizando fluxo de focagem física, mas estes dispositivos produziram emulsões com diâmetros&gt; 150 uM 9,10, cerca de uma ordem de grandeza maior do que os objectos tipicamente classificadas com FACS. Uma alternativa atractiva que incluem a funcionalidade robusta e pequena geração de gotículas de fluxo coaxial capilar de vidro de focagem com a facilidade de fabrico de PDMS litografia macia. Neste artigo, descreve um gerador de emulsão dupla que utiliza o fluxo de co-axial com foco para a produção de ≤ 50 mm emulsões e é construído inteiramente em 3D utilizando litografia macia 11. O nosso dispositivo utiliza uma abordagem de concha para fabricar dispositivos que inclui um pequeno canal de corte (Figura 1) para aproximar os processos de formação de emulsão em um bico capilar de vidro puxado. Mais importante ainda, estes dispositivos requerem nenhum tratamento de superfície específica, e toda a construção de polímero proporciona fácil e reproduzível sc fabricaçãoalable para um grande número de dispositivos duplicados. Aqui, descrevemos o projeto, fabricação e testes do gerador de emulsão dupla. Geração de emulsão dupla é mostrado para ser robusta e reproduzível para baixo para diâmetros de gotículas de 14 mm. O acoplamento de funcionalidade com a facilidade de fabrico torna este dispositivo uma opção atraente para o desenvolvimento de novas aplicações de emulsão dupla.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
	</tbody>
</table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

O gerador de emulsão dupla é constituída por um dispositivo de focagem de fluxo coaxial criado utilizando PDMS 3D fabricação (Figura 1A). A geometria permite que a formação de um jacto coaxial de três fases para ser cortada num quadrado, de 50 um x 50 um orifício, permitindo a formação de água / óleo / água emulsões duplas (Figura 1B, Figura 1C). A fase aquosa interna e a fase de óleo secundária são unidas numa junção...

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Duplo Emulsion Geração de utilizar um dispositivo de polidimetilsiloxano (PDMS) Co-Axial Flow Foco

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

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Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

15.9K visualizações.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

Vídeo: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Um dispositivo de câmara de fluxo tridimensional Novel para estudar Chemokine dirigido extravasamento de células circulantes fisiológicos sob condições de fluxo

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Bioengenharia

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Alongamento Células micropatterned num PDMS Membrana

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lipid Bilayer Vesicle Geração Usando Microfluidic jateamento

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

Formação Automated Lipid bicamada da membrana Usando um polidimetilsiloxano Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Um microcontrolador operado dispositivo para a geração de extratos líquidos de aerossol de cigarro eletrônico e fumo cigarro convencional

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Imobilização de indivíduos ao vivo Caenorhabditis elegans , usando um Chip de Microfluidic polidimetilsiloxano ultrafino com retenção de água

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Geração de ratos transgênicos usando uma abordagem vetorial lentiviral

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Geração de Condições Ambientais Dinâmicas usando um dispositivo microfluido de alto rendimento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Estimativa de Pressão Auxiliada Subharmônica com Contraste (SHAPE) Usando Imagem Ultrassonográfica com Foco na Identificação de Hipertensão Portal

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...