Este trabalho foi apoiado pelo Centro Nacional de Pesquisa de Recursos e do Centro Nacional para a Promoção da Ciência Translacional, Institutos Nacionais de Saúde, através de Grant UL1TR000124 (a FW); o Felix &amp; Mildred Yip Endowed Professorship eo Fundo Família Barnes (para DTWW), do Instituto Nacional de Pesquisa Dental e Craniofacial dos Institutos Nacionais de Saúde sob Award Número T90DE022734 (de MT). O conteúdo é da exclusiva responsabilidade dos autores e não representam, necessariamente, a posição oficial do National Institutes of Health.

1. magnética baseado no grânulo exossomo Extracção <ol><li> Pipetar uma solução bem agitada de 5 mL de micropartículas magnéticas revestidas de estreptavidina em 495 ul de solução salina tamponada com fosfato (PBS), tampão num tubo de microcentrífuga para ressuspender as esferas. Lavar e ressuspender as esferas com 500 ul de PBS três vezes usando uma cremalheira magnética. A cremalheira é uma matriz de magnetos na parte lateral de uma unidade de alojamento, que pode prender os tubos de amostra de microcentrífuga. <ol><li> Para cada lavagem, primeiro deixe-os tubos de sentar-se no rack para 1 min, e em seguida, usar uma ponteira para remover cuidadosamente o buffer sobrenadante sem perturbar as contas. </li><li> Colocar os tubos em uma cremalheira regular sem ímãs na parte lateral. Adicionar 500 ul de PBS nos tubos, e usar a pipeta para misturar a solução e grânulos juntos. Em seguida, coloque os tubos de volta na prateleira magnética para separar novamente as contas a partir da solução. </li><li> Realize esta remoção de tampão via magnetização e ressuspensão em PBS umtotal de três vezes. Isso realiza uma lavagem inicial das partículas magnéticas. </li></ol></li><li> Ressuspender as pérolas em 490 ul de tampão PBS, com o tubo colocado na porção não magnetizada da cremalheira magnética. Pipet 5 l de rato biotinilado anticorpo anti-CD63 humano a 1,0 mg / ml de concentração de ações para a mistura de grânulos. Utilizar a pipeta para misturar os grânulos e anticorpo em solução. </li><li> Inserir os tubos de microcentrífuga com rebordo e mistura de anticorpo biotinilado num rotor amostra. Defina os parâmetros rotador para o rotator amostra para rotação recíproca a 90 ° de inclinação por 5 segundos e vibrando em 5 ° por 1 segundo. Rodar os tubos de amostra da mistura de pérolas a-esses parâmetros para 30 min à temperatura ambiente. </li><li> Remover o anticorpo não ligado após conjugação. <ol><li> Após 30 minutos de rotação, à TA, coloque os tubos em volta da cremalheira magnético durante 5 min. </li><li> Executar três lavagens de grânulos através da remoção da fase líquida utilizando uma micropipeta e lava-se com 500 μ; L de PBS. Após a lavagem triplo, ressuspender as contas em 490 mL de caseína-PBS e coloque sobre a porção desmagnetizado do rack. </li></ol></li><li> Extracção exossoma utilizando esferas revestidas com anticorpo. <ol><li> Escreva em cada tubo com ID amostra alvejado. Pipetar uma amostra de 10 uL de soro ou saliva no tubo de microcentrífuga. Utilizar a pipeta para misturar a amostra e as esferas magnéticas, pipetando várias vezes. </li><li> Colocar os tubos de amostra com e anti-humanos esferas de anticorpo CD63 no rotador e rodar durante 2 horas à temperatura ambiente. Usar os mesmos parâmetros rotador como descrito no passo 1.2. </li><li> Após 2 h de amostragem rotativa, fazer uma lavagem tripla através da magnetização de grânulos separados a partir da solução, a remoção de fase líquida com micropipeta, e ressuspensão de grânulos em 500 ul de tampão Tris-HCl. Os grânulos resultantes são agora ligado aos exossomos e estão prontos para o lançamento de campo elétrico e de medição. </li></ol></li></ol> 2. Elétrica campo induzido Lançado umnd Medição de conteúdo Exosomal <ol><li> A pré-cobertura inicial de Eletrodo com GADPH Primer <ol><li> Aplicar um poço de plástico para um conjunto de eléctrodos para impedir a contaminação cruzada dos eléctrodos individuais. Para esta experiência, use um eletrodo de matriz de 16-sensor com cada eletrodo unidade na matriz constituída por um, contador, e eletrodo de referência de trabalho feito de ouro nua. </li><li> Prepare uma mistura de ações de 100 sonda nM DNA, 0,3 M KCl, e pirrol 10 mM por pipetagem reagentes de ações em um tubo com puríssima água destilada. Misture bem em vórtice. NOTA: Para este estudo, a sonda de DNA seleccionada corresponde ao gene GAPDH de referência, que é conhecida por existir dentro de exossomas. A sequência da sonda utilizada é: 5&#39;-biotina-AGGTCCACCACTGACACGTTG-3 &#39;. Use esta mistura em todos os eletrodos. </li><li> Pipetar 60 ul da mistura de sondas de ADN-monómero sobre a superfície de cada eléctrodo de ouro. Examine os eletrodos para garantir que haja uma cobertura adequada do trabalho, counter, os eléctrodos de referência e por a mistura líquida. </li><li> Electropolymerize mistura de monómero-sonda para criar uma camada de polímero condutor sobre a superfície do eléctrodo por aplicação de uma onda quadrada (CSW) cíclico perfil do campo eléctrico para a superfície do eléctrodo. Este campo elétrico consiste na aplicação de 350 mV para 9 seg e imediatamente mudar para 950 mV por 1 segundo. Aplicar este perfil cíclico onda quadrada ao eléctrodo durante 10 ciclos, para um total de 100 segundos de campo eléctrico aplicado. </li><li> Lavar superfície do sensor 3 vezes com água destilada e seca com azoto gasoso para remover o líquido da superfície do eléctrodo. Certifique-se que o líquido é adequadamente removida do eletrodo. </li></ol></li><li> Exosome Carga Descarga <ol><li> Carga 5 uL de 1 uM de uma sonda de detecção em 495 uL da mistura de complexo de talão-exossoma e usar uma pipeta para misturar. NOTA: A sonda de detecção é uma sequência de iniciador de ADN conjugado com uma molécula de fluoresceína na extremidade 3 &#39;. A prov detectore sequência utilizada para este estudo corresponde ao ARNm de GAPDH encontrado dentro de exossomas. A sequência da sonda detectora conjugada com fluoresceína é: 5&#39;-GCAGTGGGGACACGGAAGGCC-Fluoresceína-3 &#39;. </li><li> Pipeta 60 ul da sonda e mistura complexa talão-exosome em uma superfície do eletrodo de ouro com uma matriz ímã embaixo. Esta matriz ímã é constituído por dezesseis 2,54 milímetros de diâmetro de neodímio ímãs alinhados para corresponder aos eletrodos de trabalho do sensor. Figura 2A ilustra a colocação dos ímãs e solução talão-exosome. </li><li> Uma vez amostra é carregado na superfície do eletrodo, aplicar 20 ciclos de campo elétrico CSW com 9 segundos a -300 mV e 1 seg a 200 mV (200 sec total). A carga exosomal que é libertado irá hibridar com os iniciadores na superfície do eléctrodo. Se os marcadores de superfície do exosome são objeto de investigação, pule esta parte do experimento. Figura 2B ilustra esse processo. </li> &lt;li&gt; Wash-off os analitos não ligados na superfície do eletrodo por triplo enxaguar a superfície do eletrodo com água destilada. Seque o eletrodo com gás nitrogênio. </li></ol></li><li> Anticorpo repórter e Leitura <ol><li> Adicionar 60 ul de 150 unidade / anticorpo anti-fluoresceína conjugado ml de peroxidase de rábano (HRP em 1: 1000 de diluição) diluídos em caseína / PBS. </li><li> Use um campo elétrico dirigido conjugação a HRP anti-fluoresceína complexo para o sanduíche de sonda. Aplicar -200 mV durante 1 segundo e 500 mV durante 1 seg durante 5 ciclos até à superfície do eléctrodo. A Figura 2A mostra os detectores de captura e complexos de sonda, tanto para um sistema de ácido nucleico e proteína. </li><li> Superfície do sensor tríplice lavagem com água destilada e seque com gás nitrogênio. </li><li> Após a lavagem-off de desacoplado excesso de anticorpo anti-fluoresceína, adicionar 60 ul de 3,3 &#39;, 5,5&#39;-tetrametilbenzidina (TMB). Coloque esta substrato sobre a superfície de cada sensor com uma pipeta multicanal. </li><li> Realizar leitura amperométrica da corrente através da medição da corrente do eléctrodo em -200 mV durante 60 segundos usando um potenciostato electroquímico capaz de medição simultânea de 16 canais. A Figura 2C é um exemplo de perfil de corrente durante a leitura. </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/52439/52439fig2highres.jpg" /> Figura 2. Componentes de Efirm. Método (A) Método de extracção de exossomas usando biofluid CD63 anti-humano de micropartículas revestidas magnéticos e, em seguida, a descarga de carga exossoma utilizando ondas quadradas cíclicos aplicados ao complexo partícula-exossoma. (B) Esquema de eletrodo biosensor utilizado para detecção de alvos de RNA / DNA / proteína do exosome liberado. (C) Exemplo representativo de leitura amperometric da metodologia Efirm, onde maior magnitude atual corresponde to níveis mais altos de uma biomolécula. Este valor é a partir de Wei et al. 14 <a href="https://www.jove.com/files/ftp_upload/52439/52439fig2large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a>

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David Wong é co-fundador da RNAmeTRIX Inc., uma empresa de diagnóstico molecular. PeriRx LLC sublicensed propriedades intelectuais pertencentes ao diagnóstico molecular de RNAmeTRIX. David Wong é um consultor para PeriRx.

Como os resultados indicam, CD63 anti-humano revestido nanopartículas magnéticas são capazes de capturar especificamente pequenas partículas que têm um tamanho que varia 70-100 nm. Esta partícula capturado é consistente com o perfil observado anteriormente de exossomas. Além disso, o uso da CSW baixa voltagem após a captura das partículas é mostrado para removê-los da superfície do grânulo e provocar perfis de degradação de ADN semelhante ao de um método tradicional de tampão de lise com base para a li...

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>. The disclosures were updated.

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>.

Errata: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Pesquisa exosome é um campo emergente de investigação que examina microvesículas lipídicas que carregam um RNA, DNA 2, 3 e proteína de carga. Inquéritos anteriores da biologia exosome levaram a identificação de exossomos em fluidos biológicos como sangue 4, 5 urina, leite materno 6, 7 e saliva. Estudos têm demonstrado que os exossomas desempenhar um papel em diferentes vias celulares, meditar remotamente a comunicação entre diferentes sistemas do corpo 8. Devido ao papel exossomas desempenhar na comunicação intercelular, é a hipótese de que eles podem empacotar alvos biomoleculares (proteína, RNA e DNA) correlacionados com estados de doença. 3 in vitro e animal modelo 9 estudos parecem confirmar esta hipótese. Ao investigar o conteúdo exosomal para descoberta de biomarcadores, é necessário o desenvolvimento de uma metodologia para o isolamento exosome seletiva de fluidos biológicos, expulsi induzidona carga a partir de exossomas, e quantificação de biomoléculas de exossoma. No âmbito do presente trabalho, os exossomas irá ser definido como uma estrutura com um diâmetro de cerca de 70-100 nm e que possui marcador de superfície CD63. Os investigadores tipicamente primeiro purificar os exossomas por ultracentrifugação a 10 e, em seguida, processar o conteúdo exosomal através do uso de kits de tampão de lise. O uso de métodos de tampão de lise requer tempos de incubação variam de minutos a horas. Este processo pode potencialmente prejudicar carga exosome e levar para provar degradação. Por exemplo, o ARN exossoma salivar libertado através de tampão de lise para o ambiente extracelular circundante possui uma semi-vida de menos de 1 min, tornando a medição de ARN exosomal pós-tampão de lise uma tarefa particularmente difícil sem a adição de reagentes de estabilização 11. O efeito combinado da adição de vários reagentes para lise e estabilização podem introduzir agentes que complicam e interferir com a Analysis de conteúdo exosomal. Uma abordagem alternativa pode ser útil para uma rápida descarga conteúdo exosomal e preservar a segurança da carga para caracterização. Neste trabalho, propomos o uso de um campo elétrico não uniforme para a liberação de conteúdo exosomal. Elétrico-campos foram conhecidos para levar a capacidade de polarizar e perturbar a bicamada lipídica que forma as membranas celulares. Nosso trabalho experimental explora o uso de não-uniformes cíclicos ondas quadradas (CSW) para interromper a estrutura microvesículas de exossomos e liberando a carga transportada. Este método utiliza tensões na faixa de várias centenas de milivolts, o que significa que a maioria das biomoléculas não será interrompido. Nós demonstramos que o uso de uma onda quadrada-cíclico é capaz de accionar o conteúdo lançamento de ARNm salivar exossoma no ambiente circundante fluídica. Esta versão do conteúdo exosomal está perfeitamente integrado com um sistema de eletrodos que podem ser usados ​​para quantificar os níveis de expressão de biomarcadores 12,13. Este método proposto permite, e tampão de lise análise livre rápido, sensível do conteúdo exosome. <img alt="Figura 1" src="/files/ftp_upload/52439/52439fig1highres.jpg" /> Figura 1. Visão Geral do Efirm Workflow.. O método Efirm é dividida em três fases principais que são necessários para a purificação e análise de exossomos. Este método de libertação conteúdo exosomal e análise baseada CSW é usado em conjunto com microesferas magnéticas específicos de CD63 para isolamento exossoma. Estes grânulos de CD63 por afinidade para permitir o isolamento selectivo de exossomas a partir de amostras salivares (e outros fluidos biológicos). Após a incubação e extração de exossomos usando as esferas magnetizadas, as contas são migrados para o sistema de sensor eletroquímico para a CSW baseado liberação de conteúdo e análise de parte do experimento. A Figura 1 apresenta uma visão geral do trabalhofluir do método Efirm.

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			<td height="21" style="height:21px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helios 16-Channel Reader System with Chip Interface&#160;</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td style="width:132px;">&#160;RS-1000-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16X Sensor Chip, Bare Gold, pack of 5 chips</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td>SC1000-16X-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Biotinylated anti-human CD63 Antibody</td>
			<td style="width:157px;">Ancell, USA</td>
			<td style="width:132px;">215-030</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dynabeads MyOne Streptavidin T1</td>
			<td>Invitrogen, USA</td>
			<td>65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Neodynium Magnetics ( 1/10&#34; dia. x 1/32&#34; thick)</td>
			<td style="width:157px;">K&#38;J Magnetics, USA</td>
			<td>DH101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Ultrapure Distilled Water</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Mettler Toldeo 3M KcL Solution</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>1911512</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Pyrrole</td>
			<td style="width:157px;">Sigma-Aldrich, USA</td>
			<td style="width:132px;">W338605-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Anti-Fluorescein-POD, Fab fragments</td>
			<td style="width:157px;">Roche, Germany</td>
			<td>11426346910</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:347px;">3, 3&#8242;, 5, 5&#8242; tetramethylbenzidine substrate (TMB/H2O2, low activity)&#160;</td>
			<td style="width:157px;">Neogen, Usa</td>
			<td style="width:132px;">330175</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Phosphate Buffered Saline Solution</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td align="right" style="width:132px;">10010023</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Casein/PBS</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>37532</td>
			<td></td>
		</tr>
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</table>

detection of exosomal biomarker by electric field-induced release and measurement (efirm)

Exossomos são estruturas microvesicular que desempenham um papel de mediação na comunicação intercelular. É de interesse para o estudo da carga interna de exossomos para determinar se eles são portadores da doença biomarcadores discriminatórias. Para a realização de análise exosomal, é necessário desenvolver um método para a extracção e análise de exossomas a partir de fluidos biológicos alvo sem danificar o conteúdo interno. Libertação eléctrica induzida por campo e medição (Efirm) é um método para a extracção especificamente exossomas a partir de fluidos biológicos, descarregar as suas cargas, e testando o seu conteúdo de ARN / proteína interna. O uso de um anticorpo CD63 específico de micropartículas magnética anti-humana, exossomos são primeiro precipitado a partir de fluidos biológicos. Após a extracção, de baixa tensão ondas quadradas eléctricas cíclicos (CSW) são aplicados para romper a membrana vesicular e causar o descarregamento da carga. O conteúdo do exossoma é hibridado com iniciadores de ADN ou de anticorpos imobilizados numa superfície de eléctrodo para quantification de teor molecular. O método Efirm é vantajoso para a extracção de exossomas carga e descarga para análise sem tampão de lise. Este método é capaz de efectuar a detecção específica de ambos os alvos de ARN e proteína de biomarcadores na exossoma. Efirm extrai exossomas especificamente com base nos seus marcadores de superfície, em oposição a técnicas baseadas em tamanho. A microscopia electrónica de transmissão (TEM) e ensaio de demonstrar a funcionalidade do método de captura e análise de exossoma. O método foi aplicado para Efirm exosomal análise de 9 ratinhos injectados com células de pulmão humano H640 do cancro (uma linha de células transfectadas para expressar o marcador CD63 humano exossoma-GFP), a fim de testar o seu perfil contra exossoma 11 ratinhos que receberam controlos de solução salina. Níveis elevados de biomarcadores exosomal (GAPDH gene de referência e proteína de superfície do marcador CD63 humano-GFP) foi encontrado com o H640 injetaram em ratos em ambas as amostras de soro e saliva. Além disso, saliva e amostras de soro foram demonstradas ter linearidade (R = 0,79). Estes resultados são sugestivos para a viabilidade de biomarcadores de exossoma salivares para a detecção de doenças distais.

Validação de exossomo Captação de contas usando TEM Isolamento de exossomos de saliva usando CD63 esferas magnéticas anti-humanos foi validado seguindo o protocolo de extração, utilizando imagens de microscopia eletrônica de transmissão (MET). TEM mostra esferas magnéticas com grânulos 70-100 nm imediatamente adjacentes (ver Figura 3A, 3B e), de acordo com o perfil conhecido de exossomos. Não foram observados grânulos de 70-100 nm para as esfera...

Watch this Scientific Journal Video about Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM at JoVE.com

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Detecção de Exosomal Biomarker por induzida pelo campo elétrico de lançamento e Mensuração (Efirm)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

detection-exosomal-biomarker-electric-field-induced-release

Research

JoVE Journal

Bioengineering

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

18.3K visualizações.  University of California, Los Angeles. Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves. 

Vídeo: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Desenvolvimento personalizado Ovário de Hamster Chinês hospedar-Cell ensaios de proteína utilizando a Tecnologia micropartícula Acústico Membrana

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Bioengenharia

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Medição de Coesão agregados por tensiometria superfície do tecido

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

Medição da tensão Lançamento Durante Laser Induced Axon lesão axonal Avaliar Adesão ao substrato em piconewton e resolução de milissegundos

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A detecção óptica de<em&gt; E. coli</em&gt; Bactérias por mesoporosos Silicon Biossensores

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Medição do isométrica máxima força gerada pelo permeabilizadas fibras musculares esqueléticas

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Diferenciação de células precursoras neurais induzidas por campo elétrico em dispositivos microfluidos

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Modelagem de elementos finitos de um microambiente elétrico celular

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivos de campo elétrico e magnético para estimulação de tecidos biológicos

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.