O financiamento para esta pesquisa foi fornecido pela New Jersey Comissão sobre a medula espinhal Research (NJCSCR) (a FHK), conceder CSCR14IRG005 (a BLF), NIH conceder R15NS087501 (a CHC), ea Fundação Kirby FM (a ETA).

NOTA: Por favor, consulte todas as folhas de dados de segurança pertinentes (MSDS) antes do uso. Alguns dos produtos químicos utilizados no presente protocolo são tóxicos e cancerígenos. Por favor, use todas as práticas de segurança adequadas (extractor de fumo, porta-luvas) e equipamentos de proteção individual (óculos de segurança, luvas, jaleco, calça de corpo inteiro, sapatos fechados) ao utilizar materiais tóxicos ou ácido / base. 1. Fabricação de Mestre Moldes para macio Litografia usando fotolitografia <ol><li> Desenhar o layout dos microcanais, utilizando uma ferramenta de desenho assistido por computador (CAD). </li><li> Imprimir o layout em uma placa de máscara em branco usando escritor máscara laser. </li><li> Lavar uma bolacha de silício de 4 polegadas com acetona seguido de isopropanol e seco com uma pistola de ar de azoto para assegurar que não solvente é deixado sobre a bolacha. </li><li> Desidratar a bolacha cozendo-o sobre uma placa quente a 200 ° C durante 5 min. </li><li> Selecione um fotorresiste negativo projetado para a altura desejada dos microcanais. Fou exemplo, usar fotorresistente negativo SU-8 50 para obter microcanais com uma altura de 50 mm. </li><li> Permitir que a bolacha se arrefecer até à temperatura ambiente, e depois alinhar a bolacha sobre o mandril de uma máquina de revestimento de centrifugação. </li><li> Dispensar 4 mL (um diâmetro / polegada mL da bolacha) de material fotosensitivo negativo para o centro da bolacha. </li><li> Girar revestir o wafer com um ciclo de centrifugação de duas etapas usando um spinner. Em primeiro lugar, aplicam-se um ciclo de propagação de 500 rpm, com uma aceleração de 100 rotações / s para 10 s. Em seguida, aplicar um ciclo de centrifugação de 2000 rpm, com uma aceleração de 300 rotações / s para 30 s 2 para se obter um revestimento de 50 um de material fotosensitivo sobre uma bolacha. </li><li> coza macia do wafer em duas etapas em uma placa quente de acordo com as instruções do fabricante. Para um revestimento de 50 um de foto-resistente, em primeiro lugar pré-cozem a bolacha a 65 ° C durante 6 minutos e depois rampa imediatamente acima da temperatura da placa quente para o pós-coza a bolacha a 95 ° C durante 20 min. </li><li> Permitir que a bolacha se arrefecer até à temperatura ambiente e, em seguida, carregar owafer para o palco litografia. </li><li> Alinhar a máscara sobre a bolacha usando alinhador máscara e expõe a bolacha à luz UV (de acordo com as instruções do fabricante), a uma intensidade de 1,5 mW / cm2 durante 146 s de aplicar a dose total de UV de 220 mJ / cm 2 necessária para uma 50 um espessa camada de fotorresiste. </li><li> Aplicar coza pós exposição ao wafer em duas etapas em uma placa quente. Para um revestimento de 50 mícrons de material fotosensitivo, primeiro pré-cozem a bolacha a 65 ° C durante 1 min e imediatamente rampa até a temperatura da placa quente para o pós-asse a 95 ° C durante 5 min. </li><li> Desenvolver o wafer em submergindo a bolacha em SU-8 desenvolvedor e agitando até que os recursos tornam-se claras sobre o wafer. Desenvolver o wafer de ~ 6 min para o fotorresiste de espessura 50 mm. </li><li> Lavar o excesso de desenvolvedor com isopropanol e etanol, em seguida, secar o wafer com uma pistola de ar de azoto. </li><li> Disco cozer a bolacha de silício sobre uma placa quente a 150 ° C durante 15 min. </li><li> Coloque obolacha num exsicador com 25 uL do agente de silanização tridecafluoro-1,1,2,2-tetrahydrooctyl-1-triclorossilano. NOTA: O agente de silanização é corrosivo, portanto, deve ser usado em um exaustor de ácido / base, com equipamento adequado de proteção individual (óculos de segurança, luvas, jaleco, calça de corpo inteiro, fechou-toe sapatos). </li><li> Aplicar vácuo durante 5 minutos e expor a bolacha aos vapores de silano durante 30 min sem libertar o vácuo. </li><li> Transferir a bolacha em um prato de Petri de tamanho adequado. </li></ol> 2. Fabricação de Mestre Moldes para macio Litografia com fita adesiva <ol><li> Desenhar o layout dos microcanais, utilizando uma ferramenta de desenho CAD e imprimir o desenho em papel branco. </li><li> Limpar uma lâmina de vidro grande o suficiente para acomodar o projeto com isopropanol e depois secar o slide com uma pistola de ar. </li><li> Anexar fita adesiva sobre a lâmina de vidro limpa. Seja não tomar cuidado para prender as bolhas de ar. </li><li> Tape os lados do GLAss deslizar sobre o papel com o design, com o lado da fita para cima. </li><li> Usar um bisturi para cortar a fita sobre a lâmina de vidro usando o design de papel como uma referência e, em seguida, descascar fita de áreas não desejadas de a lâmina de vidro. </li><li> Lavar a lâmina de vidro com isopropanol e depois seque com uma pistola de ar. Cozer a lâmina de vidro em um forno a 65 ° C durante 30 min. </li><li> Rolar suavemente sobre a fita adesiva com um rolo de borracha para retirar as bolhas de ar e permitir que a corrediça se arrefecer até à temperatura ambiente. </li></ol> 3. Macio Litografia Fabricação dos dispositivos PDMS <ol><li> Misture PDMS elastómero e o seu agente de cura numa proporção de 10: 1 (w: w), agita-se a mistura vigorosamente, e, em seguida, desgaseificar a mistura, colocando-o em uma câmara de vácuo até que não há bolhas de ar aparecer na mistura. </li><li> Verter a mistura para um molde de silício silanizada ou molde de fita adesiva num prato de petri para obter um ~ 2 espessa camada de PDMS e desgaseificar mm até todo o ar bolhas desaparecem. </li><li> Curar o PDMS em um fornoa 65 ° C durante 2 h. </li><li> Use um bisturi para cortar a camada de PDMS pelo menos 5 mm de distância das características e, em seguida, descasque as PDMS curado fora do molde usando pinça. </li><li> Um único orifício de entrada com uma biópsia de um milímetro perfurar qualquer lugar no microcanal, de preferência ao fim de uma rede de microcanais como pode ser visto na Figura 2-A e 4-B. </li><li> Limpar o PDMS expressos com fita adesiva para remover partículas de poeira adsorvidos sobre o dispositivo. Esteriliza-se o dispositivo de microcanal PDMS (PDMS fundido) por lavagem com uma solução de etanol a 70%, seguido por estéril desionizada (DI) de água, e expor a UV durante 30 min. </li><li> Manter o dispositivo PDMS estéril numa placa de Petri estéril até à sua utilização. </li></ol> 4. Substrato Padronização <ol><li> Coloque as PDMS estéreis expressos usando uma pinça para uma lamela de vidro esterilizada ou placa de petri e aplique uma leve pressão com a ponta da pinça. NOTA: O elenco PDMS faz um conformado, mas vedação reversível com a gllamela asno ou placa de petri formando microcanais sem o uso de qualquer adesivo. </li><li> Coloque uma gota (20 - 40 ul) da solução de substrato na entrada cobrindo completamente o orifício de entrada. Padrão poli-D-lisina (PDL), colocando uma gota de 20 ul de PDL em tampão tetraborato de sódio na entrada dos microcanais. </li><li> Colocar a placa de Petri num vácuo de ~ 254 mmHgA (equivalente a uma câmara de vácuo em laboratórios biológicos) durante 10 min, e observar o ar que sai do canal sob a forma de bolhas. </li><li> Desligar o vácuo e observar a solução de substrato que flui para o microcanal. </li><li> Incubar o prato com as condições adequadas para a adesão substrato. Ver Quadros 1 e 2 para as condições de incubação (estes variam dependendo do substrato). Para padronização PDL, incubar durante 1 h a 37 ° C. </li><li> Retire cuidadosamente o selo PDMS usando uma pinça estéreis e lavar o padrão na lamela de vidro três vezes com água DI. </li&gt; <li> Adicionar uma solução a 1% de albumina de soro bovino (BSA) para a placa de petri para cobrir o prato ou a lamela de vidro e incubar durante a noite a 37 ° C. NOTA: Isto irá reduzir a aderência não específica em regiões não revestidos. </li><li> Aspirar fora a solução de BSA a 1%, e o substrato modelado está agora pronto para usar. </li></ol> 5. Padronização indireta de Células <ol><li> Preparar a suspensão de células em meio de cultura isento de soro. O tipo de célula e a densidade de células são listadas na Tabela 1. </li><li> Adicionar a suspensão de células para o prato modelado Petri e completamente imersa a região modelada em suspensão de células. </li><li> Incubar a placa de Petri a 37 ° C em uma incubadora durante 15 minutos para permitir que as células para anexar o substrato modelado. </li><li> Aspirar o excesso de suspensão de células a partir da placa de Petri e lavar as células modelado três vezes com salina tamponada com fosfato (PBS), enquanto agitando durante 10 segundos para remover células não ligadas. </li><li> Adicionar a umppropriate cultura médio para as culturas de células padronizadas e incubar as células padronizadas a 37 ° C em uma incubadora de CO 2. </li></ol> 6. Padronização direta de células NOTA: Esta técnica é uma alternativa à padronização de células indirecto descrito no passo 5. No entanto, ao contrário de no passo 5, nesta técnica células são modelados em superfícies de cultura de tecidos com ou sem revestimento de substrato. <ol><li> Esteriliza-se o dispositivo de microfluidos por lavagem com uma solução de 70% de etanol seguido por água desionizada. </li><li> Embeber o dispositivo durante a noite numa solução de 1% de BSA para evitar a adesão celular ao PDMS. </li><li> Seca-se o dispositivo à temperatura ambiente e coloque-o da parte inferior da placa de petri tratadas com cultura de tecidos. </li><li> Preparar a suspensão de células em meio de cultura isento de soro. O tipo de célula e a densidade de células são listadas na Tabela 2. </li><li> Coloque uma gota (4-8 mL) da suspensão de células, suficiente para encher omicrocanais, na entrada cobrindo completamente o orifício de entrada. </li><li> Colocar a placa de petri em vácuo durante 10 minutos e observar ar que sai do canal sob a forma de bolhas. Desligar o vácuo e observar a suspensão de células que flui para o microcanal. </li><li> Incubar a placa de Petri dentro de uma incubadora para promover a adesão celular de acordo com as condições de incubação (dependem do tipo de célula modelado) mencionado na Tabela 2. Adicionar PBS numa placa de Petri submergindo o dispositivo de PDMS. </li><li> Descasque o PDMS com cuidado para fora da placa de Petri com uma pinça e lavar o padrão com PBS. Adicionar meio de cultura de células para a placa de Petri e colocar a cultura de células na incubadora. </li></ol>

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<li>Zheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+immunoassay+through+in-channel+microfluidic+patterning%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">High-throughput immunoassay through in-channel microfluidic patterning.</a> Lab on a Chip. 12 (14), 2487-2490 (2012).</li>
<li>MacLeish, P., Barnstable, C., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro%5BTitle%5D)+AND+%22Procs+Nat+Acad+of+Sci%22%5BJournal%5D)">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Procs Nat Acad of Sci. 80 (22), 7014-7018 (1983).</li>
<li>Suchodolskis, A., et al. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/sia.3763/abstract">Elastic properties of chemically modified baker's yeast cells studied by AFM.</a> Surf Interface Anal. 43 (13), 1636-1640 (2011).</li>
</ol>

The authors declare no competing financial interests.

Enquanto fotolitografia convencional é uma técnica bem estabelecida para a realização de moldes de litografia macia, o equipamento, materiais, e as competências necessárias para utilizar fotolitografia convencional não estão prontamente disponíveis para a maioria dos laboratórios. Para laboratórios que não têm acesso a esses recursos, nós apresentamos a fabricação de fita adesiva como um método de criação de moldes com características relativamente simples para dispositivos microfluídicos. Este mét...

Em engenharia de tecidos e biosensing, a capacidade de controlar a organização espacial de proteínas e células em uma escala um, tornou-se cada vez mais importante ao longo das últimas quatro décadas 1, 2, 3. Organização espacial precisa de proteínas e células tem permitido aos pesquisadores examinar a interação entre as células e os substratos que contêm tipos semelhantes ou diferentes de células, para orientar o crescimento celular, e para imobilizar biomoléculas para a fabricação de biossensores 4, 5, 6, 7, 8, 9. Os métodos atuais de proteínas padronização incluem photopatterning e impressão microcontact. Photopatterning utiliza material sensível à luz que é reticulado por exposição a ultruma violeta (UV). Luz UV dirigida a uma fotomáscara (consistindo em áreas transparentes com regiões mais escuras para evitar a transmissão de luz UV) provoca reticulação em regiões específicas que podem então ser utilizados para a subsequente ligação de biomateriais ou células 10, 11. Embora este esquema é muito preciso e permite o controle preciso da topografia da superfície da cultura, é limitada a biomoléculas sensíveis aos raios UV, que pode ser modelado por radiação UV 12. Impressão microcontact é outro método popular de padronização proteínas específicas 13, 14. Neste método, um siloxano (PDMS) selo poli-dimetil é tratada com uma variedade de reagentes de modificação da superfície antes de ser embebido numa solução de substrato escolhido biomolecular. Em seguida, é suavemente pressionado para uma lamela de vidro ou outra superfície assim &quot;estampagem&quot; a biomolécula sobre a superfície da cultura. HoWever, estampagem se limita ao tipo de material que pode ser transferido assim como a molhabilidade de biomoléculas à superfície do PDMS selo 15. Padronização directa de células pode ser mais difícil e baseia-se em métodos de complexos, tais como substratos comutáveis, métodos com base estêncil, ou modelação com moléculas de adesão celular específicos 16, 17. Estes métodos estão limitados na sua capacidade de células de teste padrão, devido à falta de substratos de adesão celular compatíveis, a incompatibilidade do processo para o trabalho com células sensíveis biológicos e constrangimentos, inconsistência em reproduzir o padrão e a complexidade do processo. Por exemplo, substratos com comutáveis, substratos mercadorias necessitam de ser concebido para cada tipo de célula, para mudar a sua adesão a tipos específicos de células, sem a degradação por exposição à luz UV e calor usado no processo 17, &lt;<html> classe sup = &quot;xref&quot;&gt; 18, 19, 20. Estêncil métodos de padronização com base são versáteis em sua capacidade de células padrão; No entanto, é difícil fabricar matrizes PDMS nas espessuras adequadas para utilização 16, 21. injeção direta de células em canais microfluídicos PDMS tem algumas vantagens, tais como: 1) facilidade na fabricação de canais microfluídicos e 2) adequação para muitas células e diferentes substratos. No entanto, o problema predominante de captura de bolhas de ar durante o processo de injecção devido à hidrofobicidade dos PDMS sem o uso de limpeza de plasma, ou outros métodos para diminuir as bolhas de ar, faz com que seja difícil criar consistentemente células modelado sobre superfícies de vidro ou de plástico 21. Este trabalho expande micromolding capilar 22, 23,lass = &quot;refex&quot;&gt; 24, 25, 26 e relata um método para injectar suspensões de células de proteína e em microcanais. O método utilizado aqui demonstra a padronização de substratos e ambos padronização direta e indireta de tipos específicos de células. Esta técnica ultrapassa a elevada hidrofobicidade de PDMS e elimina a presença de bolhas durante a injecção quer de substratos ou células, tirando proveito da permeabilidade aos gases de PDMS 27. Este documento revela a utilização da técnica com vários substratos diferentes e tipos de células. O artigo também destaca a fabricação de moldes para litografia macia usando fotolitografia convencional, bem como um método simples e adesivo de baixo custo tape útil em recursos configurações limitadas 28, 29.

<table><tbody><tr><td>CorelDRAW X4 CAD Drawing Tools</td><td>Corel Corporation, Canada</td><td>X4 Version 14.0.0.701</td><td>CAD tool used to draw the layout of the microfluidic device</td></tr><tr><td>Laser Printer HP</td><td>Hewlett Packard, CA</td><td>1739629</td><td>Used to print the layout of microfluidic device for adhesive tape technique</td></tr><tr><td>Bel-Art Dessicator</td><td>Fisher Scientific, MA</td><td>08-594-16B</td><td>Used to degass the PDMS mixture</td></tr><tr><td>Adhesive Scotch Tape</td><td>3M Product, MN</td><td>Tape 600</td><td>Used to fabricate adhesive tape Master</td></tr><tr><td>PDMS Sylgard 184</td><td>Dow Corning, MI</td><td>1064291</td><td>Casting polymer</td></tr><tr><td>Petri Dish</td><td>Fisher Scientific, MA</td><td>08-772-23</td><td>Used to keep the mold to cast with PDMS</td></tr><tr><td>Stainless steel Scalpel (#3) with blade (# 11)</td><td>Feather Safety Razor Co. Ltd. Japan</td><td>2976#11</td><td>Used to cut the PDMS</td></tr><tr><td>Tweezers</td><td>Ted Pella, CA</td><td>5627-07</td><td>Used to handle the PDMS cast during peeling</td></tr><tr><td>Glass slides</td><td>Fisher Scientific, MA</td><td>12-546-2</td><td>Used as surface to pattern the Substrate</td></tr><tr><td>Glass slides</td><td>Fisher Scientific, MA</td><td>12-544-4</td><td>Used as surface to pattern the Substrate</td></tr><tr><td>Rubber Roller</td><td>Dick Blick Art Materials, IL</td><td>40104-1004</td><td>Used to attach adhesive tape on glass without trapping air bubbles</td></tr><tr><td>Laser Mask Writer</td><td>Heidelberg Instruments, Germany</td><td>DWL66fs</td><td>Used to fabricate quartz mask used in photolithography fabrication process</td></tr><tr><td>EVG Mask Aligner (Photolithography UV exposure tool)</td><td>EV Group, Germany</td><td>EVG 620T(B)</td><td>Used to expose the photoresist to UV light</td></tr><tr><td>Spin Coater Headway</td><td>Headway Research Inc, TX</td><td>PWM32-PS-CB15PL</td><td>Used to spin coat the photoresist on silicon wafer</td></tr><tr><td>Photoresists SU-8 50</td><td>MicroChem, MA</td><td>Y131269</td><td>Negative photoresist used for mold fabrication</td></tr><tr><td>SU-8 Devloper</td><td>MicroChem, MA</td><td>Y020100</td><td>Photoresist developer</td></tr><tr><td>Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane</td><td>UCT Specialties, PA</td><td>T2492-KG</td><td>Coat mold to avoid PDMS adhesion</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich, MO</td><td>190764</td><td>Cleaning Solvent</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich, MO</td><td>24102</td><td>Sterilization Solvent</td></tr><tr><td>Poly-D-Lysine hydrobromide (PDL)</td><td>Sigma-Aldrich, MO</td><td>P0899-10MG</td><td>PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich, MO</td><td>L2020</td><td>Laminin aliquoted into 10 &amp;micro;L aliquots and diluted to 20 &amp;micro;g/&amp;micro;L in PBS prior to use</td></tr><tr><td>BSA</td><td>Fisher Scientific, MA</td><td>BP1605100</td><td>Cell culture</td></tr><tr><td>C2C12 Myoblast cell lline</td><td>ATCC, VA</td><td>CRL-1722</td><td>Used to demonstrate C2C12 patterning</td></tr><tr><td>PC12 Cell Line</td><td>ATCC, VA</td><td>CRL-1721</td><td>Used to demonstrate PC12 patterning</td></tr><tr><td>Collagen type 1, rat tail</td><td>BD Biosciences</td><td>40236</td><td>Cell culture</td></tr><tr><td>DMEM</td><td>GIBCO, MA</td><td>11965-084</td><td>Cell culture</td></tr><tr><td>Horse Serum, heat inactivated</td><td>Fisher Scientific, MA</td><td>26050-070</td><td>Cell culture</td></tr><tr><td>Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC)</td><td>Sigma-Aldrich, MO</td><td>P1951</td><td>To label cells</td></tr><tr><td>Calcein-AM live dead cell Assay kit</td><td>Invitrogen, MA</td><td>L-3224</td><td>Cell viability Assay</td></tr><tr><td>Biopsy Hole Punch</td><td>Ted Pella, CA</td><td>15110-10</td><td>Punched hole in PDMS</td></tr></tbody></table>

a versatile method of patterning proteins and cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

Este método permite a padronização de proteínas e padronização indireta de células usando canais microfluídicos sem saída com dimensões tão pequenas quanto 10 mm e equipamentos disponíveis em quase todos os laboratórios biológicos uma vez que o molde mestre é feita. Esta técnica pode ser utilizada com canais microfluídicos PDMS criados usando fotolitografia suave tradicional, ou com canais microfluídicos PDMS criadas com fita adesiva de fabricação (Figura 1)</...

Watch this Scientific Journal Video about A Versatile Method of Patterning Proteins and Cells at JoVE.com

A Versatile Method of Patterning Proteins and Cells

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Um método versátil de padronização proteínas e células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

a-versatile-method-of-patterning-proteins-and-cells

Research

JoVE Journal

Bioengineering

9.3K visualizações.  Rutgers University. This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Vídeo: A Versatile Method of Patterning Proteins and Cells

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Bioengenharia

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Padronização de células co-cultura Usando duas fases aquosas Sistemas

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Padronização celular em photolithographically Definido Parylene-C: SiO<sub&gt; 2</sub&gt; Substratos

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

Impressão microcontact de Proteínas de Biologia Celular

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Biologia

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

Mapeando a organização espacial emergente de células de mamíferos usando Micropatterns e imagens quantitativas

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Biologia do Desenvolvimento

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Micropatterning Transmissão Electron Microscopy Grids para Posicionamento Direto celular dentro de fluxos de trabalho de tomografia crio-elétron inteira

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Bioquímica

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Preparação de Microambientes de Matriz Extracelular Incapaz para avaliar especificação de fenótipo celular schwann

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Padronização simples, acessível e modular de células usando DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Controle da Geometria Celular através de Micropatterning Assistido por Laser Infravermelho

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Geração de Padrões para Microscopia de Tração de Micropattern

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...