Esta pesquisa foi apoiada pelo programa de pesquisa de ciência básica através da nacional Research Foundation de Coreia (NRF) financiado pelo Ministério da ciência, TIC e futuro planejamento (NRF-2014R1A1A2057527 e 2016R1D1A1B03934418-NRF).

1. preparação do através de-furo alumínio placa e ímã matriz
<ol><li>Preparar duas 50 mm x 50 mm (ou mais) placas de alumínio. A espessura de cada placa foi 300 µm que é metade do diâmetro do grânulo.</li>
	<li>Forma uma matriz do através de-furo de 30 x 30 em uma das placas de alumínio usando um gravador giratório do CNC com uma Φ550-µm micro broca com 30 mm/s de taxa de mergulho e 8000 RPM de velocidade do eixo. A distância entre cada buraco (centro) foi de 1 mm (Figura 1a e Figura 2a, eu).</li>
	<li>Forma uma matriz de 30 x 30 de Φ750-µm através de buracos na outra chapa de alumínio, usando o mesmo procedimento como descrito em 1.2 (Figura 1a e Figura 2a, ii).</li>
	<li>Anexar as duas placas uns aos outros, usando uma fita adesiva e formar Φ3 furos de alinhamento mm em cada um dos quatro cantos de ambas as placas de alumínio.</li>
	<li>Mergulhe as placas de alumínio em 15% ácido sulfúrico para 12h limpar suas superfícies. Desde que a fina camada de óxido de alumínio sobre a superfície do alumínio torná-lo resistente à corrosão, o diâmetro do furo e da espessura da placa não são alterados por este tratamento com ácido.</li>
	<li>Forma uma matriz de 30 × 30 de 1 x 1 x 1 ímãs de neodímio mm (com uma força magnética de 0.363 N). Certifique-se de que cada ímã é a polaridade oposta a seu vizinho. Para evitar a quebra ou a dispersão da matriz de ímã, anexe uma placa de alumínio de 30 x 30 mm para o fundo da matriz ímã usando fita dupla-face (Figura 2a, iii e baixo-relevo na Figura 2).</li>
</ol>2. processo de captura do grânulo
<ol><li>Alinhar e empilhar as duas chapas de alumínio (top placa: 750-µm-buraco placa, placa inferior: 550-µm-buraco placa) usando os furos de alinhamento preparado nos quatro cantos de cada placa (Figura 1b).</li>
	<li>Bloquear as duas placas juntas inserindo parafusos M3 os furos de alinhamento e fixe os parafusos com porcas (Figura 1b).</li>
	<li>Empilhe o conjunto de placa de alumínio na matriz de ímã preparado (Figura 1b, 2be 2C). Alinhe a matriz de ímãs e a matriz de através de orifícios na placa de alumínio durante o processo de integracao. Em seguida, use uma fita adesiva para fixar a posição da matriz de ímã.</li>
	<li>Lugar, um número suficiente de Φ600 mm SUJ2 aço de pérolas sobre o conjunto de placa e manipular as contas usando um acryl (metálicos ou não) da placa tal que um grânulo se torna prendido em cada furo (Figura 1C, 1De 1e), enquanto simultaneamente removendo os excesso grânulos que não tenham apresentado nos buracos.</li>
	<li>Remova cuidadosamente a placa superior para evitar dispersão indesejado e luxação dos grânulos presos (Figura 1f).</li>
</ol>3. côncavo de microplacas fabricação
<ol><li>Mova o molde côncavo de microplacas, produzido em passos 2.1 a 2.5, acima, para uma placa de Petri.</li>
	<li>Misture o monômero polydimethylsiloxane (PDMS) e agente de cura, de acordo com instruções19 as indicações do fabricante com um monômero PDMS: relação de 10:1 do agente de cura.</li>
	<li>De gás da mistura PDMS usando um dessecador e bomba de vácuo para remover quaisquer bolhas presas na mistura de PDMS.</li>
	<li>Despeje a mistura PDMS no molde côncavo de microplacas e de gás novamente usando o mesmo procedimento como descrito no ponto 3.3 (Figura 1f).</li>
	<li>Asse a mistura PDMS em uma chapa de fogão a 80 ° C por 2 h formar um substrato PDMS grânulo-incorporado (Figura 1G).</li>
	<li>Remova o substrato PDMS curado do molde (Figura 1G). No processo de remoção, pulverize metanol usando garrafa de lavagem para retirar o substrato PDMS do molde.</li>
	<li>Usando um Φ15 x ímã de 2 mm, remova as esferas de aço presas ao substrato PDMS (Figura 1-h). Para este processo, pode ser usado qualquer ímã suficientemente forte para extrair os grânulos de substrato PDMS.</li>
</ol>4. cultura de esferoide
<ol><li>Corte o substrato PDMS côncavo de microplacas-modelados usando um soco de biópsia Φ14 mm deve ser montado em placa de 24 neste estudo.</li>
	<li>Esterilize o substrato PDMS de mm Φ14 resultante em um esterilizador autoclave a 121 ° C e 15 libras por polegada quadrada.</li>
	<li>Coloque o substrato esterilizado de PDMS em um prato bem 24.</li>
	<li>Revesti o substrato PDMS inteiro com 4% (p/v) pluronic F-127 solução durante a noite para evitar o acessório de celular para a superfície de microplacas. Durante o processo de revestimento, remova quaisquer bolhas de ar aprisionadas no côncavos micropoços pipetando ou usando um líquido de limpeza ultra-sônico.</li>
	<li>Liberar a solução de F-127 três vezes usando tampão fosfato salino (PBS).</li>
	<li>Sementes de 1 mL de solução de meio-celular (de Dulbecco meio Eagle modificado) (que contém 2 x 106 células) sobre o substrato PDMS. Observe que a densidade de semeadura pode ser alterada de acordo com o tamanho do esferoide de destino e/ou tipo de célula de destino. Aqui, células-tronco adiposo-derivado (ASC) foram utilizadas.</li>
	<li>Aspire a 1 mL de meio usando uma 1000 µ l pipetas para remover qualquer excesso células que não foram presos aos micropoços (Figura 3).</li>
	<li>Incube as celulas a 36,5 ° C, umidade de > 95% e 5% CO2 condição. No caso dos ASCs usados em nosso estudo, as células agregam-se um spheroid em 48 h.</li>
</ol>

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Os autores têm sem conflitos de interesse a divulgar.

O grande desafio deste método de fabricação foi a fixação segura dos grânulos da matriz do através de-furo na placa de alumínio. Para resolver este desafio, força magnética sob a forma de uma matriz de 30 x 30 imã foi usada para corrigir os grânulos firmemente, como mostrado nas figuras 6 e 7. A densidade do fluxo magnético da matriz de ímã, que tem a polaridade oposta, é mais forte no centro de cada superfície do ímã. Porque a força da força magnética depende da de...

As células cultivadas em forma de esferoide são mais semelhantes ao tecido real no corpo que uma cultura planar bidimensional1. Dada esta vantagem, o uso de esferoides tem sido adotado para melhorar o estudo da interação de célula para célula2,3, ativação imune4,5e de diferenciação6de despistagem de drogas. Além disso, incorporando vários tipos de células de esferoides recentemente foram aplicados ao organoids (perto de-fisiológicos tridimensional (3D) tecido), que são muito úteis para o estudo de desenvolvimento e doença humana7. Vários métodos têm sido utilizados para produzir esferoides. O método mais simples envolve a utilização de uma superfície não-adesivas, tais que as células agregam-se com os outros e esferoides de formulário. Um prato de Petri pode ser tratada com albumina de soro bovino, pluronic F-127 ou um polímero hidrofóbico (por exemplo, metacrilato de 2-hydroxyethl de poli) tornar-se sua superfície não adesivas,89. O método de girador-balão é outro meio conhecido de produzir grandes quantidades de esferoides10,11. Neste método, as células são mantidas em suspensão por mexendo para impedi-los de tornar-se ligado ao substrato. Em vez disso, o flutuante células agregado de esferoides de formulário. O método de superfície não-adesivas e o girador balão método podem produzir grandes quantidades de esferoides. No entanto, eles estão sujeitos a limitações, incluindo dificuldades em controlar o tamanho de esferoide, bem como o acompanhamento e monitoramento de cada esferoide. Como um remédio para esses problemas, um outro método de produção de esferoide, ou seja, a suspensão drop método pode ser empregado12. Isto envolve depositando gotas de suspensão de células na parte inferior da tampa de um prato de cultura. Estas gotas são geralmente 15 a 30 µ l de tamanho e contêm cerca de 300 a 3000 células13. Quando a tampa é invertida, as gotas são mantidas no lugar pela tensão superficial. O ambiente de microgravidade em cada gota concentra as células, que então formam esferoides único em uma interface líquido-ar livre. Os benefícios do enforcamento método drop são que oferece uma distribuição de tamanho bem controlados, enquanto é fácil de rastrear e monitorar cada esferoide, em relação os métodos de balão de superfície e spinner não adesivas. No entanto, esse método incorre em uma desvantagem em que a produção em massa de esferoides e o próprio processo de produção é excessivamente do trabalho intensivo.
Uma matriz de microplacas é um flat placa com muitos poços de tamanho micro, cada um com um diâmetro que varia de 100 a 1000 µm. O princípio de produção de esferoide quando usando micropoços é semelhante do método de superfície não-adesivas. Os benefícios incluem o fato de que os poços da microplaca fornecem espaços entre os poços da microplaca para separar as células ou esferoides, tal que é fácil de controlar o tamanho de esferoide, enquanto também facilitando a monitorar cada único esferoide. Com um grande número de poços da microplaca, produção de alto rendimento esferoide também é possível. Outra vantagem do micropoços é a opção para poços de formulário de diferentes formas (hexahedral, cilíndrico, trigonal prismáticos) dependendo fins experimentais originais dos usuários. Geralmente, no entanto, uma forma tridimensional (3D) côncava (ou hemisférica) é considerada como sendo o mais adequado para a produção de esferoides único tamanho uniforme. Portanto, a utilidade de micropoços côncavos tem sido relatada para muitos estudos de biologia celular tais como aqueles examinando a casos de diferenciação de células-tronco embrionárias14, a secreção de insulina das células da ilhota clusters de15, o atividade enzimática de hepatócitos16e a resistência de droga de tumor esferoides17.
Infelizmente, a fabricação de micropoços frequentemente requer instalações especializadas micropatterning; métodos baseados em fotolitos convencionais requerem exposição e instalações em desenvolvimento enquanto métodos reativos baseados em íon-gravura precisam de equipamento de plasma e de feixes de iões. Esse equipamento é caro, que, juntamente com o processo de fabricação complicada, apresenta uma alta barreira à entrada para os biólogos que não têm acesso a microtecnologia. Para superar estes problemas, outros métodos de baixo custo tais como gelo litografia18 (usando as gotas de água congelada) e o método de membrana flexível14 (usando uma membrana, substrato do através de-furo e vácuo) têm sido sugeridos. No entanto, esses métodos também incorrer em graves inconvenientes tais como sendo difícil controlar os tamanhos padrão, a obtenção de elevadas proporções e a produção dos poços da microplaca área maior.
Para superar as questões acima, propomos um método de fabricação do romance de microplacas côncavo utilizando um substrato do através de-furo, esferas de aço e uma matriz de ímã. Usando esse método, centenas de micropoços esféricos côncavos podem ser fabricadas, aproveitando o mecanismo de força magnética-assistida de travamento automático grânulos metálicos (Figura 1). O processo de fabricação envolve o uso de muito poucas facilidades caros e complicados e não exige muitas habilidades avançadas. Como tal, pessoas mesmo não qualificadas podem facilmente realizar este método de fabricação. Para demonstrar o método proposto, humanos adiposo-derivado de células-tronco foram cultivadas aos micropoços côncavos para produzir esferoides.

<table><tbody><tr><td>CNC rotary engraver</td><td>Roland DGA</td><td>EGX-350</td><td></td></tr><tr><td>Micro drill bit</td><td>HAM Pr&amp;auml;zision</td><td>30-1301 TA</td><td>&amp;Phi; 0.55 and 0.75 mm</td></tr><tr><td>Sulfuric acid 98%</td><td>Daejung</td><td>7683-4100</td><td>For cleaning aluminum plate.&lt;br/&gt; Dilute with distilled water with 15% solution</td></tr><tr><td>Neodymium magnet</td><td>Supermagnete</td><td>W-01-N</td><td>1 x 1 x 1 mm</td></tr><tr><td>Bearing ball</td><td>Agami Modeling</td><td>SUJ2</td><td>&amp;Phi; 600 &amp;mu;m steel bead</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dowcorning</td><td>Sylgard 184</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma Aldrich</td><td>p2443</td><td>Dilute with phosphate buffered saline to 4% (w/v) solution</td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (D-PBS)</td><td>ATCC</td><td>30-2200</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Adipose-derived mesenchymal stem cells</td><td>ATCC</td><td>ATCC PCS-500-011</td><td></td></tr></tbody></table>

a paired bead and magnet array for molding microwells with variable concave geometries

Uma cultura de esferoide é uma ferramenta útil para compreender o comportamento celular em que proporciona um na vivo-como ambiente tridimensional. Vários métodos de produção de esferoide como superfícies não adesivas, frascos de girador, gotas de suspensão e micropoços têm sido utilizados em estudos de interação célula a célula, ativação imune, drogas, triagem, haste diferenciação celular e geração de organoides. Entre esses métodos, os poços da microplaca com uma geometria tridimensional côncavo ganhou a atenção de cientistas e engenheiros, dados suas vantagens de geração de esferoide de tamanho uniforme e a facilidade com que as respostas de esferoides individuais podem ser monitorado. Apesar de cost-effective métodos tais como o uso de membranas flexíveis e litografia de gelo têm sido propostos, estas técnicas incorrer em graves inconvenientes tais como dificuldade em controlar os tamanhos padrão, realização de altas proporções e produção de áreas maiores de micropoços. Para superar estes problemas, propomos um método robusto para fabricar micropoços côncavos, sem a necessidade de instalações complexas de alto custo. Este método utiliza uma matriz de através de-furo 30x30, aço de cem micrômetro-ordem vários grânulos e a força magnética para fabricar 900 micropoços em um substrato de polidimetilsiloxano (PDMS) 3 x 3 cm. Para demonstrar a aplicabilidade do nosso método para aplicações biológicas de células, nós cultivadas as células-tronco adiposas por 3 dias e produzido com sucesso usando a nossa plataforma de microplacas de esferoides. Além disso, realizamos uma simulação magnetostatic para investigar o mecanismo, pelo qual força magnética foi usada para aprisionar os grânulos aço os através de buracos. Acreditamos que o método de fabricação de microplacas proposto poderia ser aplicado para muitos estudos baseados em esferoide celulares como triagem de drogas, regeneração de tecidos, diferenciação de células-tronco e metástases.

Um molde convexo e padrão de microplacas com êxito foram fabricadas seguindo as etapas 2.1 a 3.7. (Figura 4). As esferas de aço comerciais ficaram presos na matriz do através de-furo de 30 x 30. Os grânulos realizaram-se, firmemente, sem quaisquer lacunas entre os grânulos e os correspondentes através de furos (figura 4a). A forma de fabricados de microplacas côncava é côncavo hemisférica, com um diâmetro de 600 µm, ...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Este manuscrito introduz um método robusto de fabricar micropoços côncavos, sem a necessidade de instalações complexas de alto custo. Usando a força magnética, esferas de aço e uma matriz do através de-furo, várias centenas de micropoços formaram-se em um substrato de polidimetilsiloxano (PDMS) 3 x 3 cm.

Um grânulo emparelhado e matriz de ímã para moldagem micropoços com geometrias côncavas variáveis

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

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Universidad de Cartagena UDEC

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8.6K visualizações.  Chung-Ang University. Este manuscrito introduz um método robusto de fabricar micropoços côncavos, sem a necessidade de instalações complexas de alto custo. Usando a força magnética, esferas de aço e uma matriz do através de-furo, várias centenas de micropoços formaram-se em um substrato de polidimetilsiloxano (PDMS) 3 x 3 cm.

Vídeo: A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Fabricação de microbolhas de Côncavo-porosidade Beads PDMS

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Química

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive.
Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

We show a simple and rapid method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development.

Shrinky Dink-Hanging Drops: Uma maneira simples para formar e Cultura corpos embrióides

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

Biologia

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Criação de Sub-50 nm nanofluidic Junções em PDMS Microfluidic Chip via processo de automontagem de coloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Engenharia

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Formação de biomembrana Microarrays com um método de montagem baseada em Rodo

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengenharia

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

Polimérico Fabrication matriz Microneedle por fotolitografia

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Fácil e precisa Mecano-profiling em micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e Montagem de microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfresagem de Controle Numérico Computacional de um Dispositivo Acrílico Microfluídico com uma Restrição Escalonada para Imunoensaios Magnéticos Baseados em Nanoppartículas

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Pinças magnéticas de alta velocidade para medições nanomecânicas em elementos sensíveis à força

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Bioquímica

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabricação de uma matriz de microambiente celular Artificial multiplexado

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...