Esta pesquisa foi financiada pelo Instituto Nacional de números de subsídios Saúde R01NS050452 e R01EB009429. Fabricação dos chips cantilever foi realizada externamente por colaboradores no Centro de Nanofabricação localizado na Universidade de Cornell. Todos os equipamentos utilizados no processo de fabricação cantilever foi localizado nesta instalação. Um agradecimento especial a Mandy Esch e Jean-Matthieu Prot para a sua assistência com cantilever micro-fabricação. Animação de vídeo de funcionalidade cantilever foi gerada por Charles Hughes, Alex Zelenin e Eric Imperiale do Lab Realidade Sintética na UCF.

1. Cantilever Chip Fabrication Pormenores ilustrados das etapas de fabricação descritos são proporcionados na Figura 1. <ol><li> Coloque wafers de silício sobre isolante (SOI) em um forno e leve ao forno a 125 ° C por 20 min para desidratar-los. </li><li> Deposite um 1,5 um espessa camada de óxido de silício na camada alça do wafer SOI desidratado usando um Plasma avançado Chemical Vapor Deposition (PECVD) ferramenta. </li><li> Coloque a bolacha sobre a rotação do mandril com a camada de revestimento do dispositivo virada para cima. Certifique-se de bolacha é centrado, dispensar 2 ml de P20 iniciador no centro da bolacha, e centrifugação a 3000 rpm durante 60 segundos a uma aceleração de 1000 rpm / seg. P20 primário promove a aderência de material fotosensitivo que a camada do dispositivo. </li><li> Enquanto a bolacha ainda está no revestidor mandril rotação, dispensar 2 ml de S1818 fotorresistente no centro da bolacha, e centrifugação a 3000 rpm durante 60 segundos a uma aceleração de 1000 rpm / seg para obtain uma camada de 1,5 um de espessura de material fotosensitivo. </li><li> Coloque a bolacha sobre uma placa quente e realizar um cozimento suave a 115 ° C durante 1 min. </li><li> Realizar uma exposição UV contato duro usando um contato fotolitografia máscara alinhador, a fim de transferir o padrão da máscara cantilever para o fotorresiste sobre a camada de dispositivo. Calcule o tempo de exposição com base no valor da energia de exposição de 125 mJ / cm 2 para S1818 photoresist. </li><li> Desenvolver o photoresist imergindo o wafer em um 726 MIF desenvolvedor photoresist para 1 min, agitando suavemente o wafer e para trás. </li><li> Lavar a bolacha com (DI) de água ionizada-de e secá-lo, de preferência, usando um secador de rotação da ferramenta de enxaguamento (SRD). </li><li> Remover o material fotoresistivo a partir da borda da pastilha (até 5 mm a partir da extremidade), utilizando um cotonete de algodão embebido em acetona. </li><li> Coloque o wafer em uma ferramenta profunda Reactive Ion Etch (DRIE), com o lado da photoresist voltada para cima, e executar uma receita para gravar a camada de silício modelada através do devicamada ce. As funções da camada de óxido enterrado como uma parada de etch, por isso, realizar uma etch com 50% a 100% mais ciclos do que se espera que seja necessário para garantir a corrosão completa. </li><li> Coloque a bolacha em uma solução de banho photoresist quente por 20 minutos para retirar o fotorresiste. Transfira a bolacha para uma quick-dump-enxaguadora (QDR) para lavar o wafer com água DI. Após a tira photoresist, coloque o wafer em uma ferramenta SRD para lavar e secar o wafer. Inspecione o wafer sob um microscópio para garantir o padrão cantilever aparece como esperado. </li><li> Deposite um 1 mícron espessa camada de óxido de silício dopado com un na camada do wafer usando uma ferramenta de PECVD dispositivo. Funções desta camada de óxido como uma camada protetora para as consolas durante mais etapas de processamento. </li><li> Coloque a bolacha sobre a rotação do mandril com o revestimento de camada punho virada para cima para iniciar o processamento do lado posterior do disco. Certifique-se de bolacha é centrado, dispensar 2 ml de P20 iniciador no centro da bolacha, e centrifugação a 3000 rpmpor 60 segundos a uma aceleração de 1000 rpm / seg. </li><li> Enquanto a bolacha ainda está no revestidor mandril rotação, dispensar 2 ml de SPR220-4.5 fotorresistência sobre o centro da pastilha, e centrifugação a 2000 rpm durante 45 segundos a uma aceleração de 1000 rpm / seg para se obter um 6,5 uM camada espessa de photoresist. </li><li> Coloque a bolacha em uma placa quente para efectuar uma aproximação suave cozer a 115 ° C durante 2 min. </li><li> Usando um fotolitografia contato alinhador capazes de frente / trás alinhamento, alinhar a máscara de janela traseira para o padrão cantilever frente e realizar uma exposição UV contato duro para transferir o padrão da máscara de janela traseira do fotorresiste sobre a camada de alça. Use um tempo de exposição calculado com base no valor da energia de exposição de 480 mJ / cm 2 para SPR220-4.5 photoresist. </li><li> Após a exposição à radiação UV, deixar as bolachas permanecer numa área escura durante 30 minutos antes do próximo passo de processamento. </li><li> Desenvolver o fotorresistente por imersão a bolacha emum desenvolvedor 726 MIF photoresist por 2 min, agitando suavemente o wafer e para trás. </li><li> Lavar a bolacha com (DI) de água ionizada-de e secá-lo, de preferência, usando um secador de rotação da ferramenta de enxaguamento (SRD). </li><li> Coloque as bolachas em uma estufa a 90 ° C durante 12 hr. </li><li> Etch a camada de óxido de silício sobre a parte traseira da bolacha usando um sistema de RIE com gases fluorados e uma receita para gravar óxido. O óxido modelado na parte traseira da pastilha actua como uma máscara de corrosão para o tratamento posterior. Inspeccionar as bolachas sob um microscópio para assegurar que o óxido de silício na janela é exposta completamente gravado. </li><li> Remover o material fotoresistivo sobre a borda da pastilha (até 5 mm a partir da extremidade), utilizando um cotonete de sala limpo embebido em acetona. </li><li> Coloque a bolacha numa ferramenta com DRIE e executar uma receita para gravar a camada de alça modelada a uma profundidade de 500 mm com o funcionamento enterrado camada de óxido como um batente de corrosão. Dividir este etch em várias execuções para evitar um aquecimento excessivo dabolacha, que provoca corrosão inconsistente do silício. Esta etapa etch remove completamente o silício por baixo das consolas. </li><li> Executar um passo de ataque químico húmido utilizando 25% de produto corrosivo HF diluído, para retirar a camada de óxido enterrado abaixo dos braços de suporte e a camada de óxido de protecção na parte superior dos braços de suporte. NOTA: Este passo liberta a superfície de fundo dos braços de suporte de silício e também abre uma janela de baixo para fornecer um acesso para sondar o cantilever com o laser. </li><li> Lavar a bolacha usando uma série de banhos de água DI e cuidadosamente seca com azoto. Uma vez que os cantilevers são suportados apenas em suas bases, não use sprays fortes de água DI ou gás inerte directamente nas consolas. </li><li> Clivar as fichas individuais a partir da bolacha com as linhas clivam produzidos durante o passo de pega camada de corrosão. </li></ol> Cultura 2. celular <ol><li> Prepare 13F lamelas de acordo com métodos previamente publicados 17. NOTA: Se 13F enseadarslips não estão disponíveis, qualquer superfície hidrofóbica com um ângulo de contacto acima de 95 ° pode ser usado. </li><li> Esterilizar fichas cantilever e 13F lamelas numa solução de etanol a 70% e deixar secar ao ar numa câmara de fluxo. </li><li> Coloque fichas cantilever individuais no alto de 13F lamelas dentro de um padrão de 12 poços. </li><li> Revestir as consolas com o biopolímero ou modificação superficial optimizada para o tipo de célula a ser utilizado de acordo com os protocolos de cultura de células padrão. </li><li> Re-suspender as células no seu meio de crescimento específica para a concentração desejada. NOTA: Este protocolo resulta em um grande número de células que caem através da janela do cantilever e que não aderem à superfície cantilever desejado. Preparações de células deve, portanto, ser feita 3-4x mais concentrado do que para os preparativos lamela normal. Por exemplo, a sementeira de células satélites do músculo esquelético de rato é tipicamente levada a cabo usando uma densidade de sementeira de aproximadamente 500 a 700 células / mm quadrado 15,16, E são utilizados com substratos cantilever a 2.000 células / mm quadrado. Experiências de optimização deve ser levada a cabo com as novas fontes de células para determinar uma densidade de sementeira apropriada. </li><li> Pipete 200 pi da suspensão de células sobre a superfície do chip de cantilever, garantindo a bolha de forma cobre as janelas totalmente cantilever. Se o meio de mechas através da janela e não forma uma bolha estática na parte superior dos braços de suporte substituir a lamela 13F e Reattempt o chapeamento. </li><li> Transferir a placa contendo as fichas para uma incubadora e permitir que as células a aderir durante pelo menos 1 hora (preferencialmente 2-3 h). </li><li> Após este período de chapeamento, utilizar uma pinça estéril para transferir o chip a um bem limpo, sem uma lamela 13F, e superior a cultura com 1 ml de meio de crescimento. </li><li> Retorne a placa para a incubadora. </li><li> Manter as células de acordo com o seu protocolo padrão para a manutenção in vitro em lamelas. Para as células de músculo esquelético,um interruptor de composição do meio que induz a formação de miotubo uma vez as células se tornavam confluentes será necessário. </li></ol> 3 Configuração de Hardware e Software de Análise cantilever deflexão <ol><li> Coloque um prato de cultura para a fase de aquecimento de um microscópio vertical electrofisiologia. </li><li> Adicionar 3 ml do meio de alimentação das células encontra-se suspensa em (+ HEPES 10 mM) para a fase de microscópio aquecida. </li><li> Montar os eléctrodos de aço inoxidável sobre o interior da placa de cultura aquecido a uma distância de 15 mm, e ligá-los a um gerador de impulsos, capaz de produzir impulsos de estimulação do campo de intensidade variável, da frequência e da forma de onda, para permitir que o sistema para produzir estimulação do campo de células, quando apropriado. </li><li> Parafuso de um laser de hélio-néon, montado em fases de translação XY, para o lado de baixo da mesa de microscópio e ajustá-lo de modo que o feixe de laser é dirigida através da base do prato de cultura aquecido a um ângulo de 30 ° relativE para o plano do cantilever. </li><li> Tranque um módulo fotodetector quadrante, montado em estágios de translação XY, ao lado de baixo do palco microscópio e ajustar a posição para que as terras de feixe de laser refletido no centro dos quatro quadrantes. Figura 2 apresenta uma visão geral da configuração do hardware necessário para a implementação do protocolo descrito. </li><li> Escreva um programa de software para controlar os atuadores lineares que fazem a varredura através das consolas. Escrever o programa de software, com referência ao diagrama de fluxo representado na Figura 3. A interface gráfica programado para utilização com este sistema é fornecida na Figura 4. </li></ol> 4 Gravação cantilever deflexão de dados <ol><li> Ligue o hardware e software de análise cantilever associado. </li><li> Insira o termistor fase aquecida para o meio e esperar por ele para ler 37 ° C. </li><li> Insira o chip cantilever no palco com a cantilevers voltadas para o lado direito do palco. </li><li> Ligue a fonte de luz do microscópio. </li><li> Concentre o microscópio para trazer as extremidades dos braços de suporte em vista e usar o software de controlo do laser / foto-detector para posicionar o feixe de laser na extremidade do braço de suporte 1 NOTA: Assumindo que os braços de suporte estão orientadas para a direita do palco, um cantilever é aquele posicionado no canto superior esquerdo da matriz e os números de correr para 16 no canto inferior esquerdo. Cantilever 17 está na posição superior direito e é executado a 32 na parte inferior direita (Figura 5A). </li><li> Pressione o botão &quot;Play&quot; no software de gravação. </li><li> Posicione a foto-detector para que o sinal lê &quot;0&quot; em ambos x e y quadros, ajustando os motores de passo que controlam o fotodetector. </li><li> Jogo 1 em cantilever posição no software de controlo do laser / fotodetector (Figura 4). </li><li> Mova o laser até a ponta do cantilever 16, repita o passo 4.7., E definir cantilever 16 posição no software de controle do laser / foto-detector. </li><li> Mova o laser até a ponta do cantilever 32, repita o passo 4.7., E definir cantilever 32 posição no software de controle do laser / foto-detector. </li><li> Mova o laser até a ponta do cantilever 17, repita o passo 4.7., E definir cantilever 17 posição no software de controle do laser / foto-detector. </li><li> Desligue a fonte de luz do microscópio e a luz do teto no laboratório. </li><li> Pressione o botão &quot;record&quot; no software de gravação. </li><li> Defina o hardware gerador de pulso de 40 ms, 5 V pulsos com uma frequência de 1 Hz, e ligar a máquina. NOTA: Opcionalmente, empregam protocolos de estimulação otimizadas para fontes de células específicas, neste ponto, as definições indicados são orientações baseadas em dados coletados através de fontes humanas e células de roedores 12,13,15,16. </li><li> Usando o software de controle do laser / fotodetector, definir o hardware para fazer a varredura em toda a matriz de 32 cantilever, parando por 5 segundos a cada um eme. </li><li> Quando a digitalização dos 32 consolas está completa, desligue o estimulador, em seguida, parar o software de gravação e abrir o arquivo de dados. </li><li> Examine o rastreamento gravado a partir de cada cantilever para a evidência de atividade contrátil. Tome nota de cada cantilever com respostas positivas. A contracção é definido como um pico se a deformação é, pelo menos, 0,1 V, acima da linha de base. </li><li> Remova todos os cantilevers não-responsivos a partir do protocolo de digitalização no software de controle do laser / foto-detector. </li><li> As consolas activas podem então ser varrida novamente, sem estimulação, a fim de obter uma leitura da actividade contráctil espontânea da célula. </li><li> Varreduras executado com ou sem campo amplo estimulação eléctrica, a seguir à adição de um composto terapêutico para o meio, a fim de observar o seu efeito sobre a saída funcional das células cultivadas. </li><li> Realizar avaliação de fadiga ao estimular eletricamente as células por longos períodos e níveis de análise de contr<html> actility para medir quanto tempo leva para o pico de força para cair abaixo de um limite específico. </li><li> Em experimentos onde os neurônios motores são mantidos em co-cultura com o músculo, medir a formação junção neuromuscular através de um tratamento de motoneuron-miotubo cantilever co-culturas com um estimulante neuronal (como o glutamato) ou inibidor sináptico (por exemplo, D-tubocurarina) e digitalização para aumentos e reduções na atividade espontânea, respectivamente 16. </li><li> Realizar exames específicos como ditado pelas necessidades dos experimentos planejados. </li></ol> 5 Análise de Dados cantilever deflexão <ol><li> Utilização modificado equações de Stoney 15 (descrito abaixo) para converter os dados da linha suspensa em bruto de deflexão (em volts) em um visor de tensão na camada de células ou miotubos (em Pascal), e uma medição directa da força contráctil celular (em nano-Newtons): ad / 51866 / 51866eq1.jpg &quot;/&gt; Equação 1 <img alt="Equação 2" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" /> Equação 2 </li><li> Onde δ = cantilever ponta tensões de deflexão e produzido pela miotubo, σ c = tensão produzida pelo miotubo, assumindo um filme de espessura uniforme a toda a largura do braço de suporte, C = o coeficiente de detector específico do sistema de tensão relativa à posição do laser sobre a foto -detector, θ = o ângulo do laser e do detector em relação ao plano do cantilever, Si = E o módulo de elasticidade de silício, Si = t a espessura do braço de suporte, t f = espessura miotubo, v = proporção de Si de veneno silício, L = comprimento do cantilever, comprimento P = caminho do laser de ponta cantilever ao detector, e w <sub&gt; Si = a largura do cantilever. Um diagrama esquemático que explica os termos utilizados nessas equações é fornecido na Figura 6. </li><li> Assumindo que o miotubo é um filme uniforme, a força no miotubo é igual à força no filme, levando a Equação 3, igualando o cálculo da força de tensão e área de secção do cruzamento célula assumiu que foi utilizado para a aplicação de Stoney de equação. <img alt="Equação 3" fo:content-width="1.5in" src="files/ftp_upload/51866/51866eq3.jpg" /> Equação 3 </li><li> Após a coleta de dados funcionais, corrigir fichas cantilever para análise imunocitoquímica ou utilizar células de proteína ou DNA análise usando técnicas convencionais. </li><li> Opcionalmente, as células voltem para a incubadora, em vez de se preparar para a análise molecular, a fim de reavaliar o desempenho funcional em um momento posterior de ponto. </li></ol>

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W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc:+Sensory+neuron+innervation+of+intrafusal+muscle+fibers.">Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers.</a> Biomaterials. 31, 8218-8227 (2010).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+tissue+engineering:+A+maturation+model+promoting+long-term+survival+of+myotubes,+structural+development+of+the+excitation-contraction+coupling+apparatus+and+neonatal+myosin+heavy+chain+expression.">Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression.</a> Biomaterials. 30, 5392-5402 (2009).</li><li>Das, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+novel+serum-free+cell+culture+model+of+skeletal+muscle+differentiation+by+systematically+studying+the+role+of+different+growth+factors+in+myotube+formation.">Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.</a> In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).</li><li>Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture.">Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.</a> Brain research. 630, 136-147 (1993).</li><li>Hickman, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry.">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., &amp; Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).</li><li>Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system.">Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system.</a> Biotechnology progress. 19, 1756-1761 (2003).</li><li>Rumsey, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Node+of+Ranvier+formation+on+motor+neurons+in+vitro.">Node of Ranvier formation on motor neurons in vitro.</a> Biomaterials. 30, 3567-3572 (2009).</li><li>Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li><li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li><li>Varghese, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643 (2010).</li><li>Natarajan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li><li>Davis, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li><li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2 (2013).</li></ol>

The authors have nothing to disclose.

Os passos críticos na análise de microescala consolas para a evidência de contracção celular são o posicionamento do braço de suporte do chip dentro da platina do microscópio e do subsequente alinhamento do laser e foto-detector, com a ponta dos braços de suporte de canto na matriz. Se isso não for feito de forma precisa, em seguida, o software não será capaz de extrapolar as posições das restantes consolas na matriz, potencialmente levando ao acúmulo de falsos negativos durante a coleta de dados. Os oper...

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipettes and tips</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

O desenvolvimento de ensaios in vitro preditivos e mais biologicamente relevantes baseia-se o avanço dos sistemas de cultura de células versáteis que facilitam a avaliação funcional das células semeadas. Para esse efeito, a tecnologia cantilever microescala oferece uma plataforma com a qual a medir a funcionalidade contráctil de uma gama de tipos de células, incluindo esquelético, cardíaco e células de músculo liso, através da avaliação da contracção induzida substrato flexão. Aplicação de matrizes cantilever multiplexados fornece os meios para o desenvolvimento moderado a protocolos de alto rendimento para a avaliação da eficácia do fármaco e toxicidade, o fenótipo da doença e progressão, assim como neuromusculares e outras interacções célula-célula. Este manuscrito apresenta pormenores para a fabricação de matrizes de cantiléver fiáveis ​​para este fim, e os métodos necessários para células em cultura com sucesso sobre estas superfícies. Descrição mais detalhada é fornecida sobre as medidas necessárias para realizar anal funcionalysis de tipos de células contrátil mantida em tais matrizes, utilizando um novo sistema de laser e foto-detector. Os dados representativos fornecidos destaca a precisão e a natureza reprodutível da análise da função contráctil possível usar este sistema, bem como a grande variedade de estudos para que tal tecnologia pode ser aplicada. Ampla adoção bem sucedida deste sistema poderia fornecer aos investigadores os meios para realizar estudos de baixo custo rápidas e funcionais in vitro, levando a previsões mais precisas do desempenho do tecido, o desenvolvimento da doença ea resposta ao tratamento terapêutico romance.

Cultura bem sucedida de células contrácteis em cantiléver é um procedimento relativamente simples, utilizando técnicas padrão de cultura de células (Figura 5). A percentagem de consolas de apoio células contratantes irá variar dependendo do tipo de célula que está sendo examinado e técnica de cultura específica empregada. Usando células embrionárias primárias derivadas de rato membros posteriores, actividade contráctil foi detectada em 12% das consolas analisados ​​(n = 4). Análise...

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Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Este protocolo descreve o uso de braços de suporte de silicone em micro-como as superfícies de cultura maleáveis ​​para a medição da contractilidade de células do músculo in vitro. Contração celular provoca flexão cantilever, que pode ser medido, registrado, e convertido em leituras de força, proporcionando um sistema não invasivo e escalável para medir a função contrátil in vitro.

Utilização de microscópicos de silício Cantilevers para avaliar a função celular contrátil<em&gt; In Vitro</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

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Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

10.1K visualizações.  University of Central Florida.  Este protocolo descreve o uso de braços de suporte de silicone em micro-como as superfícies de cultura maleáveis ​​para a medição da contractilidade de células do músculo in vitro. Contração celular provoca flexão cantilever, que pode ser medido, registrado, e convertido em leituras de força, proporcionando um sistema não invasivo e escalável para medir a função contrátil in vitro. 

Vídeo: Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

Utilização de Nanoestruturas Cristal plasmônicos e Fotônica para Manipulação de Micro-e Nanopartículas melhorada

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Bioengenharia

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Em Assembléia in vitro de Semi-artificial máquina molecular e sua utilização para detecção de danos no DNA

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantificação da função global diastólica por análise baseada em Modelagem cinemática de transmitral fluxo através do formalismo de enchimento parametrizada diastólica

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analisando falta de homogeneidade de mistura em um dispositivo micro, pela técnica Microscale Schlieren

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Técnicas para a Evolução da levedura Pentose-fermentação robusta para bioconversão de transformação de lignocelulose em etanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

Medição e Modelagem contrátil que secam no Estrato Córneo Humano

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

A<em&gt; In Vitro</em&gt; Modelo Cultura Órgão da Murino Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelos avançados de fígado 3D para testes de genotoxicidade in vitro após exposição nanomaterial de longo prazo

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.