This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. fotomáscara Fabrication <ol><li> Limpar uma bolacha de 4 &quot;de vidro com solução piranha (H 2 SO 4 / H 2 O 2 na razão 2: 1) durante 20 min a 120 ° C por imersão num tanque de quartzo. </li><li> Depositar uma camada de crómio / ouro (30 nm de Cr / 1 uM de Au) camada sobre a bolacha de vidro utilizando um feixe electrónico de evaporação 7 (Figura 1A). <ol><li> Coloque as bolachas em um evaporador e-beam. Uma vez que o vácuo atinge 5 x 10 -6 Torr, ligue a fonte de alta tensão (10 kV). Controlar a espessura pelo painel de controle monitor. </li><li> Pré-limpos cada material durante 30 segundos utilizando a pistola de feixe electrónico, mantendo o obturador &quot;OFF&quot; (a fim de evitar a deposição sobre as bolachas). </li></ol></li><li> Gerar uma camada de máscara photoresist Cr / Au de profunda corrosão úmida do vidro. <ol><li> Aplicar um fotorresistente espessura de 2 um por fiação de 5 ml da solução durante 30 segundos a 3000 rpm utilizando um sistema de revestimento por pulverização de rotação.Prebake o fotorresistente numa placa de aquecimento a 100 ° C durante 1,5 min. </li><li> Expor e asse difícil o photoresist a 120 ° C durante 30 minutos em um prato quente. É crítico para gerar uma superfície hidróf oba e uma forte aderência do foto-resistente para a camada de metal. Padrão da camada de Cr / Au Au usando Cr e condicionadores através do photoresist mascarar 8,9. </li></ol></li><li> Para a proteção da superfície do vidro modelado-un, unir temporariamente a pastilha de vidro de uma pastilha de silício manequim. 9 <ol><li> Coloque a bolacha de vidro sobre uma chapa quente a 110 ° C e a cera derreter no lado oposto da pastilha de vidro (de tal forma que toda a superfície da bolacha é coberto com cera). </li><li> Coloque uma pastilha de silício manequim em contato com a pastilha de vidro e pressione para remover o excesso de cera. A fim de evitar o derramamento de cera, coloque um lenço de papel sala limpa sobre a placa. </li></ol></li><li> Executar ataque químico isotrópico da lente usando ácido fluorídrico optimizada (49% v / v)e solução de ácido clorídrico (37% v / v) (numa proporção volumétrica de 10: 1). com um agitador magnético durante 10 min 8,5 Presença do HCl é crítico na obtenção de uma boa qualidade de superfície das lentes geradas. <ol><li> Assegure-se que a velocidade de gravação é de 7 um / min; utilizando um volume total de 200 ml de solução de gravação. Executar ataque químico num recipiente de plástico e tomar precauções de segurança para esta etapa de processamento. </li><li> Limpar a bolacha na (DI) de água desionizada por enxaguamento e secagem adicional à temperatura ambiente. </li></ol></li><li> Após a conclusão do processo, separar a pastilha de vidro a partir da bolacha de silício do manequim e aquecer a cera usando uma chapa quente a 100 ° C durante 15 seg. Como a cera derrete a esta temperatura, separar a pastilha de vidro a partir da bolacha de silício do manequim. </li><li> Remova a cera restante, o fotorresistente e as salientes Cr / Au camadas nos bordos das lentes que utilizam ultra-sons durante 1 hora, utilizando N-metil-2-pirrolidona como solvente a 80 ° C num tanque de ultra-sons.</li><li> Criar uma réplica molde PDMS das microlentes fabricadas nas máscaras 11. </li><li> Caracterizar as dimensões fotomáscara (comprimento e largura) e o molde microlentes PDMS (profundidade e diâmetro) réplicas usando um microscópio eletrônico de varredura e microscópio estereoscópico, respectivamente. 12-14 </li></ol> 2. Eixos MN Fabrication <ol><li> Criar uma cavidade de 2,5 cm x 0,9 cm, utilizando as lâminas de vidro montados em ambos os lados de um vidro. O número das lâminas de vidro empilhadas em ambos os lados vai determinar a altura da cavidade conhecido como espaçador de espessura (Figura 1B). </li><li> Fixe cada camada da placa de vidro através da aplicação de uma fina camada da solução de pré-polímero que contém o poli (etileno-glicol) diacrilato (PEGDA, PM = 258 Da) com 0,5% w / w de 2-hidroxi-2-metil-propiofenona (HMP) para a lâmina de vidro, seguido por irradiação da luz configurado com o ultravioleta de alta intensidade (UV) de 2 seg. </li><li> Posicione o photomaSK (previamente fabricado) com as superfícies de Cr / Au revestida voltada para o interior da cavidade. Assegure-se que os lados das paredes da cavidade são não obscurecer as lentes embutidas na fotomáscara. </li><li> Encher a cavidade com a solução de pré-polímero até que a superfície revestida a Cr / Au está em contacto com a solução sem quaisquer bolhas visíveis. </li><li> Irradiar a configuração com elevada intensidade de luz UV de uma intensidade desejada por 1 segundo a uma distância de 3,5 cm da fonte de UV usando a estação de cura por UV com uma gama de filtro de UV de 320-500 nm. Utilize um adaptador collimating com a sonda de luz UV. </li><li> Medir a intensidade de luz UV utilizado usando um radiómetro. </li><li> Após exposição à radiação UV, remover a foto-mascara com a matriz de milhões. Verter a solução de pré-polímero em excesso que não é polimerizado no processo volta para o seu recipiente original para reutilização. </li><li> Quantificar o diâmetro e o comprimento da ponta dos MNS usando um microscópio estereoscópico de acordo com as instruções do fabricante. </li></ol><pclass = &quot;jove_title&quot;&gt; 3. MN Backing Camada de Fabricação <ol><li> Com uma pinça, colocar os MNS (previamente fabricado) fixos sobre a foto-mascara em um poço de uma placa de 24 poços como mostrado na Figura 1C. </li><li> Adicionar um volume específico (300 - 400 ul) da solução de pré-polímero no poço, até as agulhas estão submersas a uma altura desejada. Este volume determina a espessura da camada de suporte resultante. </li><li> Irradiar a configuração com luz UV de alta intensidade (15,1 W / cm 2), 10,5 cm de distância da fonte de UV com a duração de 1 segundo. </li><li> Separa-se a camada de suporte sobre a matriz de manganês a partir da fotomáscara usando uma lâmina afiada. </li><li> Quantificar o diâmetro comprimento, diâmetro da ponta e da base do MNS com a camada de suporte usando um microscópio estereoscópico de acordo com as instruções do fabricante. </li></ol>

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The authors declare no conflict of Interest.

O protocolo acima descrito para o fabrico da matriz de MNS foi apresentada para fabricar a matriz MNS de ~ 1 cm 2. As matrizes podem ser aumentados através da criação de uma cavidade grande tamanho e usando uma fotomáscara maior. O aumento de tamanho da cavidade pode ser criado através do aumento da largura entre os espaçadores de cada lado. Embora cada passo para fabricar as matrizes MN no protocolo foi importante, os passos mais importantes foram: o posicionamento photomask, o preenchimento de solução de pré-polímero, e irradiação da configuração. Posicionamento da fotomáscara deve ser de tal maneira que as superfícies de Cr / Au revestidos enfrentar o interior da cavidade e os lados das paredes da cavidade obscurecer as lentes embutidas na fotomáscara. Ao preencher o molde com a solução de pré-polímero, garantir que o ar bolhas não são aprisionados, que pode de outra forma levar a uma matriz força MN deformado e baixo. As bolhas de ar podem ser evitados através de uma ação wicking controlada adicionando lentamente o prepolymer solução e assegurar que não existem bolhas de ar presentes na solução de pré-polímero. Posicionamento da instalação para a irradiação deve ser feito de uma forma orientada para garantir a exposição UV uniforme. Antes da exposição à luz UV, a configuração foi alinhado e colocado dentro da demarcação no suporte da base. Os MNS formadas usando fotolitografia foram grandemente influenciadas pela presença dos micro-lentes como a lente resultou na polimerização num percurso convergente, o que levou à formação dos MNS nítidas em relação aos MNS cilíndrica formada usando a foto-mascara planar. No fotomáscara planar, a luz UV passa por ele com pouco desvio (quase recta), resultando na formação dos MNS cilíndricos com pontas afiadas menos. Enquanto na fotomáscara microlente-embedded, a luz UV que passa através das lentes foram submetidos a refracção e convergentes, resultando na formação de MNS com pontas afiadas. A equação do fabricante de lente que tem sido utilizado como um predictive modelo para aproximar o comprimento do MN, correlacionando com a distância focal das microlentes deu uma previsão do comprimento três vezes menos do que o real. A discrepância pode ser devido à superfície convexa achatada das microlentes, o que não permitiu refração da luz como a lente convexa convencional 13. Outro factor para a geometria MN foi a intensidade da luz UV. A intensidade de 6,44 W / cm2 foi escolhido porque as agulhas produzidas nesta intensidade possuía uma resistência mecânica suficiente para aplicações de pele. Outra constatação foi relacionada com a intensidade com que cada incremento na intensidade da radiação UV, o comprimento de manganês aumenta. Isto poderia ser atribuído ao topo plana de microlente, o que permitiu que alguns dos raios de luz a viajar para além do ponto focal. 14,15 Além disso, a extensão da polimerização tem o seu limite, dependendo da lei do inverso do quadrado da luz, ou seja, a luz perde energia como a distânciaA partir da fonte aumenta. 16 Os protocolos aqui descritos têm a vantagem de fabricação livre de molde dentro de um curto período de tempo. Mas não podemos prever o quão longe ela pode ser levada adiante para a fabricação em massa. Os MNs matrizes foram feitas a partir de um baixo custo polímero biocompatível. Ele pode potencialmente ser usado como dispositivo de distribuição transdérmica de fármacos para aplicações farmacêuticas e cosméticas. Mais interessante, que podem ser utilizados como uma combinação de sistema de entrega e o dispositivo, tal como os compostos terapêuticos pode ser encapsulada (ou por mistura em solução de pré-polímero de solubilização) durante o fabrico de mns. 5,6,17 A solubilidade do composto acrescentado interior deve ser tomada em consideração a solução de pré-polímero como as características de MN, por exemplo, força MN, pode mudar. 17

Administração transdérmica de fármacos oferece uma abordagem alternativa atractiva para a administração da droga, especialmente para biomoléculas, que são administrados quase exclusivamente por injecções hipodérmicas. No entanto, a pele, especialmente a camada de topo (estrato córneo), é uma barreira formidável prevenção moléculas exógenas de entrar no corpo humano. Recentemente, dispositivos MN surgiram como possibilitando ferramentas para entregar drogas através da pele. Os dispositivos MN criar poros temporários no interior do estrato córneo, para permitir a passagem das moléculas de fármaco para conseguir a desejada actividade fisiológica melhorada com a adesão do paciente e conveniência 1-3. Foram adotados vários métodos de fabricação para fabricar MNs poliméricos 4. No entanto, eles geralmente envolvem processos complicados e que exigem vários passos longos tempos e / ou temperaturas elevadas para fabricar matrizes milhões. 4 Para simplificar o processo de fabricação, um único processo livre de mofo etapa usandouma fotomáscara foi desenvolvido recentemente 5,6. No entanto, com este método, fabricado MNs teve blunt dicas de agulhas, como nenhum mecanismo estava no local para modificar o caminho ultravioleta (UV) luz envolvido em fotolitografia. Neste estudo, as microlentes incorporado na fotomáscara têm sido propostos para definir a geometria dos milhões. O protocolo para fabricar máscaras que consistem em microlentes embutidos e, posteriormente, MN fabricação com pontas afiadas usando o photomask são relatados.

<table><tbody><tr><td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td><td>SIGMA&amp;nbsp;</td><td>475629-500ML</td><td></td></tr><tr><td>2-hydroxy-2-methyl-propiophenone (HMP)</td><td>SIGMA&amp;nbsp;</td><td>405655-50ML</td><td></td></tr><tr><td>Bovine collagen type 1, FITC conjugate&amp;nbsp;</td><td>SIGMA&amp;nbsp;</td><td>C4361</td><td></td></tr><tr><td>UV curing station&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>OmniCure S2000-XL</td><td></td></tr><tr><td>Collimating Adaptor&amp;nbsp;</td><td>EXFO Photonic Solutions Inc., Canada</td><td>EXFO 810-00042</td><td></td></tr><tr><td>24-well plate</td><td>Thermo Fisher Scientific, USA</td><td></td><td></td></tr><tr><td>Nikon SMZ 1500 stereomicroscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Dillon GL-500 digital force gauge&amp;nbsp;</td><td>Dillon, USA</td><td></td><td></td></tr><tr><td>A-1R confocal microscope&amp;nbsp;</td><td>Nikon, Japan</td><td></td><td></td></tr></tbody></table>

polymeric microneedle array fabrication by photolithography

Este manuscrito descreve a fabricação de microagulhas polimérico (Mn) matrizes por fotolitografia. Trata-se de um processo livre de mofo simples usando um photomask constituído por micro-lentes embutidas. Micro-lentes embutidas foram encontrados para influenciar MN geometria (nitidez). MN matrizes robustos com diâmetros que variam entre 41,5 ponta mm ± 8,4 mm e 71,6 mm ± 13,7 mm, com dois comprimentos diferentes (1336 mm ± 193 mm e 957 mm ± 171 mm) foram confeccionados. Estas matrizes de MN podem proporcionar aplicações potenciais na entrega de agentes terapêuticos de baixo molecular e macromolecular através da pele.

A geometria das MNS pode ser significativamente afectada pelas características de fotomáscara de microlentes e embutidos. O grau de refracção afecta o caminho de transmissão dos raios UV, que influenciaram a geometria MN (Figura 2A). Cada microlente foi encontrada para ter um diâmetro de 350 um, a 130 uM achatada superfície convexa, e uma profundidade de 62,3 uM (Figura 2B-D). Utilizando o teorema de Pitágoras, o raio de curvatura da primeira superfície mostrou-se 272,89 uM. A distância focal foi calculada como sendo de 509,28 pM (considerando n = 1,53627 vidro; n ar = 1,000; λ = 365 nm) através da equação fabricante de lentes 12, tal como indicado abaixo: 1 / f = (n 1 / n m -1) * (1 / r 1 -1 / r 2) Onde n é 1 o índice de refracção do material da lente, n m é o índice de refracção do meio ambiente, o símbolo R1 representa o radius de curvatura da primeira superfície, e R2 é o raio de curvatura da segunda superfície. O efeito de intensidade da radiação UV no comprimento MN, nitidez e deformação estrutural foi estudado variando a intensidade da luz UV a partir de 3.14 para 15.1 W / cm 2, a uma distância focal constante e à distância de fonte de luz. Verificou-se que o comprimento médio MN aumentou significativamente (p &lt;0,05) com o aumento da intensidade 3,14-9,58 W / cm 2 (Figura 3A). Outros aumentos de intensidade superior a 15,1 W / cm 2 não produziu alteração significativa no comprimento. O diâmetro da ponta (medida da agudeza) e a estrutura de ponta de MN foram encontrados para variar com o aumento da intensidade (Figura 3B). Os de MNS com uma forma regular e sem qualquer deformação estrutural foram observados a 6,4 W / cm2. A camada de suporte foi fabricado para permitir a remoção de manganês sob a forma de um emplastroe para tornar o reutilizável photomask. Ele também forneceu força para os eixos do manganês. Portanto, o efeito do volume de camada de suporte (o volume das soluções de pré-polímero para formar a camada posterior) foi também estudada. Com diâmetros de ponta não afectadas, foram observados os de MNS com uma gama de comprimento (1.336 ± 193 mm para 300 ul e 957 ± 171 mm para 400 ul), após a exposição à radiação UV (Figura 4). <img alt="figura 1" src="/files/ftp_upload/52914/52914fig1highres.jpg" width="700" /> Figura 1. (A) Representação esquemática do processo de fabrico de lentes embutidas fotomáscara. (1) 4 &quot;bolacha de vidro. (2) Cr / Au camada depositada utilizando um evaporador e-beam. (3) Exposição de Cr camada de máscara / Au / photoresist à luz UV com photomask. (4) Formação de teste padrão na camada usando Cr / Au etchant. (5) ligação temporária de vidro em uma pastilha de silício manequim. (6) - (7) Gravura Wet (isotrópico) processo usando HF condicionadores / HCl seguido de ultra-som. (8) descolagem de wafer de silício manequim e remoção de camada foto-resistente. (B) Representação esquemática do processo de fabrico de agulhas. Crómio fotomáscara revestido (9 x 9 matrizes), é colocado sobre uma cavidade que contém a solução de pré-polímero e exposto a UV. (C) Representação esquemática do processo de fabricação da camada de suporte. Photomask, com microagulhas em anexo, é colocado em um poço cheio com pré-polímero e exposto a UV. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52914/52914fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52914/52914fig2highres.jpg" width="450" /> Figura 2. Caracterização de fotomáscara. (A) Exposição UV foca a luz num conica<html> l caminho, produzindo MNS cónicos. (B) e uma imagem SEM de uma microlente (C). (D) uma porção de uma matriz de PDMS réplicas molde copiados dos microlentes, mostrando a superfície convexa achatada, sob um estereomicroscópio. (E) A photomask mostrando o padrão. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52914/52914fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52914/52914fig3highres.jpg" width="450" /> Figura 3. Efeito de parâmetros de UV na geometria microagulhas. Efeito de (A) e intensidade (B) espaçador espessura da duração microagulhas. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52914/52914fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> tenda &quot;fo: manter-together.within-page =&quot; always &quot;&gt; <img alt="Figura 4" src="/files/ftp_upload/52914/52914fig4highres.jpg" width="700" /> Figura 4. Efeito da variação de volume pré-polímero utilizado para a camada de suporte de fabricação. (A - B) imagens em vários tamanhos, com duração média de MN (1.336 um) MNs curtas (957 mm) e longas. (C - D) As imagens correspondentes a (AB) após o teste de força fratura. (E) Redução de comprimento MN com aumento do volume usado para camada de suporte de fabricação. Força fratura (F) MN entre os dois volumes pré-polímero utilizado para fabricar camada de suporte (BL). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52914/52914fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

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 ...

polymeric-microneedle-array-fabrication-by-photolithography

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

12.0K visualizações.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Vídeo: Polymeric Microneedle Array Fabrication by Photolithography

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Micropunching Litografia de Geração de Micro e submicrométricas-padrões sobre substratos poliméricos

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

Engenharia

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabricação de hidrogéis micropatterned para Sistemas de Cultura Neural usando fotolitografia Projeção Dinâmica Máscara

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Bioengenharia

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays de polímeros para Discovery High Throughput de Biomateriais

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

Prescrito 3-D Direto Escrita de Suspenso Micron / Sub-micron estruturas de fibras Escala através de um sistema distribuidor Robotic

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Sensor de microagulhas baseado oco para Multiplexed Transdermal Eletroquímica Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropadronização simples de células únicas sem litografia usando estêncil cortado a laser

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Biologia do Desenvolvimento

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 ...

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

Fabricação de eletrodos bem na ponta da agulha hipodérmica usando revestimento de pulverizador fotorresiste e Fotomáscara flexível para aplicações biomédicas

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Neurociência

Aplicações de impressão 3D para dispositivos de cultura de células neuronais em microescala

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Autor em destaque: Redes neuronais modulares para analisar funções cerebrais

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Polimerização de dois fótons Impressão 3D de dispositivos de cultura de células neuronais em microescala

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...