Este trabalho foi financiado em parte pelo Conselho Nacional de Ciência Taiwan grants- NSC 99-2320-B-007-005-MY2 (CC) e NSC 101-2628-E-007-011-MY3 (CMC), e do Veterans General Hospitais e University System of Taiwan Programa Comum de Investigação (CC).

Um diagrama geral do procedimento de operação é fornecida na Figura 1. Usando as práticas éticas, foram coletadas amostras de sangue de indivíduos saudáveis, e obteve amostras de humor aquoso de pacientes através do Taichung Veterans General Hospital (TCVGH), Taichung, Taiwan sob IRB aprovou protocolos ( IRB TCVGH No. CF11213-1). 1. fabricação de dispositivos de papel <ol><li> Cortar papel de cromatografia em círculos de 5 mm de diâmetro para fornecer o mesmo layout como uma microplaca de 96 poços. Sanduíche estas peças de papel com duas folhas de poliestireno com registada furos de passagem, e laminado. </li><li> Modificar dispositivos de papel, utilizando o método de conjugação química descrita nas etapas seguintes 28. <ol><li> Tratar de papel com dispositivos de plasma de oxigénio (100 mW, 1% de oxigénio, 30 seg) em uma câmara de plasma. ATENÇÃO: cuidado especial deve ser tomado quando se trabalha com trimetoxissilano 3-mercaptopropil e N -γ-maleimidobutiriloxi suecinimidaéster (GMBS), uma vez que ambos são sensíveis à humidade e tóxico. Usar luvas de protecção e evitar a inalação ou contato com a pele e os olhos. Mantenha o frasco bem fechado estoque e permitir que atinjam a temperatura ambiente antes de abrir para evitar a condensação de água. Como alternativa, abra a garrafa de dentro de um saco de luva cheia de azoto ou caixa. Alíquota em pequenos frascos para evitar a abertura freqüente da garrafa estoque. </li><li> Imediatamente incubar tratada dispositivos de papel em um (v / v) solução de 4% de 3-mercaptopropil trimetoxi silano, em etanol (200 proof) durante 30 min. </li><li> Lavar dispositivos de papel com etanol e incubar com 0,01 umol / ml GMBS em etanol durante 15 min. </li><li> Lavar com etanol (200 proof) e incubar dispositivos de papel com 10 ug / ml de avidina em solução salina tamponada com fosfato (PBS) durante 1 hora a 4 ° C. Armazenar a 4 ° C, se necessário, e usar dentro de 4 semanas. </li></ol></li><li> Molhar cada zona de ensaio de papel com 10 ul de PBS contendo 1% (w / v) de albumina de soro bovino (BSA) Por 3 × 10 min antes de manchar 10 jul biotinilado moléculas de captura por 3 × 10 min. Utilizam anti-CD63 anticorpo (20 ug / ml em PBS contendo 1% (w / v) de BSA e 0,09% (w / v) de azida de sódio) ou anexina V (1:20, v / v em tampão de ligação da anexina V) como moléculas de captura. </li><li> Enxaguar anticorpo ou anexina V moléculas anti-CD63 não ligados utilizando 10 ul correspondente PBS contendo 1% (w / v) de BSA ou anexina V soluções tampão de ligação para 3 x 1 min, respectivamente. </li></ol> 2. Serum e Aqueous Humor Coleta e Processamento <ol><li> Recolha de soro. <ol><li> Colete 10 ml de sangue periférico por punção venosa em tubos de separação de soro e inverter cuidadosamente o tubo de cinco vezes. Defina o tubo na posição vertical e aguarde 30 min. </li><li> Centrifugar a 1.200 x g durante 15 min. Transferir o soro a partir da camada superior para um tubo limpo, e centrifuga-se novamente a 3000 × g durante 30 min. </li><li> Passe o sobrenadante com um fil 0,8 mmter. Manter a amostra a -80 ° C até à sua utilização. </li></ol></li><li> Coleção humor aquoso: coletar amostras de humor aquoso de pacientes diagnosticados por um oftalmologista diretamente através de procedimentos e armazenar amostras invasiva a -80 ° C até o uso. </li></ol> 3. Isolamento de Extracelular Vesículas <ol><li> Amostras pontuais em cada zona de teste de papel a uma taxa de 5 mL / min. </li><li> Enxaguar os VE não ligados com 10 ul correspondentes PBS contendo 1% (w / v) BSA a ligação de anexina V ou soluções tampão para 3 x 1 min para dispositivos papel funcionalizados com anti-CD63 ou anticorpos anexina V moléculas, respectivamente. Execute as seguintes ensaios a jusante. </li></ol> 4. jusante Ensaio Exemplo 1: eletromicrografias de digitalização <ol><li> Corrigir EVs capturadas em funcionalizado zona de teste de papel utilizando 10 mL de 0,5 × fixador de Karnovsky para 10 min. </li><li> Lavar as amostras com amplo PBS por 2 × 5 min. </li><li> Desidratar oamostras com subsequente etanol 35% durante 10 min, de etanol a 50% durante 2 x 10 min, etanol a 70% durante 2 x 10 min, etanol a 95% durante 2 × 10 minutos, e 100% de etanol durante 4 x 10 min. </li><li> Critical seco e por pulverização catódica revestir as amostras com paládio / ouro, e examinar utilizando um microscópio eletrônico de varredura operado em baixa tensão de aceleração de elétrons (~ 5 kV). </li></ol> 5. Downstream Ensaio Exemplo 2: ELISA baseado no Livro <ol><li> Adicionar uma solução contendo 5 uL de anti-CD9 anticorpo primário a 1: 1000 em PBS a cada zona de ensaio contendo VE capturados. Espere 1 min. </li><li> Lavar as amostras com 30 ml de PBS foram agitados a 100 rpm durante 30 segundos. </li><li> Adicionar 5 uL de uma solução contendo peroxidase de rábano (HRP) -ligada anticorpo secundário em 1: 1000 em PBS e esperar 1 min. </li><li> Lavar as amostras com 30 ml de PBS foram agitados a 100 rpm durante 30 segundos. </li><li> Adicionar um substrato colorimétrico 5 ul contendo 1: 1 razão em volume de peróxido de hidrogénio umad 3,3 &#39;, 5,5&#39;-tetrametilbenzidina (TMB) e digitalizar utilizando um scanner de mesa. </li></ol> 6. Ensaio jusante Exemplo 3: Isolamento de ARN <ol><li> Mergulhar as amostras em 35 uL de auxílio de isolamento de ARN baseada em polivinilpirrolidona e 265 ul de lise / tampão de ligação para lisar os VE capturados. Vortex vigorosamente. </li><li> Adicionar 30 ul de homogenato fornecido no kit de isolamento ao lisado. Vortex vigorosamente e colocar em gelo durante 10 min. </li><li> Extrai-se o ARN utilizando a separação do ácido fenol-clorofórmio descrito nas etapas seguintes. <ol><li> Tome 330 ul de fenol-clorofórmio a partir da camada do fundo do frasco e adiciona-se ao homogeneizado / lisado. Vortex vigorosamente. </li><li> Centrifuga-se a 10000 xg durante 5 min. Transferir a fase aquosa (superior) para um tubo limpo e anotar o volume removido. </li></ol></li><li> Precipitar o ARN total com etanol a 100% do volume que é 1,25 vezes o volume da fase aquosa removida na etapa anterior e collect o ARN na solução de eluição pré-aquecido a 95 ° C. </li><li> Concentrar e purificar o ARN utilizando um kit de limpeza de ARN de acordo com o protocolo do fabricante. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

Os passos mais críticos para o isolamento bem sucedido de subgrupos de vesículas extracelulares são: 1) uma boa escolha de papel; 2) uma cobertura estável e elevada de moléculas de captura na superfície das fibras de papel; 3) manuseio adequado de amostras; e 4) práticas de higiene geral de laboratório. Os materiais porosos têm sido utilizados em muitos ensaios de baixo custo e sem equipamento. Eles podem ter o tamanho de poro sintonizável, funcionalidade versátil, baixo custo e a...

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.
EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met. 
We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chromatography Paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3001-861</td>
			<td>&#160;Whatman&#174; Grade 1 cellulose paper</td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl) trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>175617</td>
			<td>This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818</td>
			<td>Absolute, 200 Proof, molecular biology grade</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>BioShop Canada Inc.</td>
			<td>ALB001</td>
			<td>Often referred to as Cohn fraction V.</td>
		</tr>
		<tr>
			<td>N-g-maleimidobutyryloxy succinimide ester (GMBS)</td>
			<td>Pierce Biotechnology</td>
			<td>22309</td>
			<td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Pierce Biotechnology</td>
			<td>31000</td>
			<td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td>
		</tr>
		<tr>
			<td>Biotinylated mouse anti-human anti-CD63</td>
			<td>Ancell</td>
			<td>215-030</td>
			<td>clone AHN16.1/46-4-5</td>
		</tr>
		<tr>
			<td>biotinylated annexin V</td>
			<td>BD Biosciences</td>
			<td>556418</td>
			<td>Annxin V has a high affinity for phosphotidylserine (PS)</td>
		</tr>
		<tr>
			<td>Primary anti-CD9 and secondary antibody</td>
			<td>System Biosciences</td>
			<td>EXOAB-CD9A-1</td>
			<td>The secondary antibody is horseradish peroxidise-conjugated</td>
		</tr>
		<tr>
			<td>Serum separation tubes</td>
			<td>BD Biosciences</td>
			<td>367991</td>
			<td>Clot activator and gel for serum separation</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer</td>
			<td>BD Biosciences</td>
			<td>556454</td>
			<td>10X; dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>TMB substrate reagent set</td>
			<td>BD Biosciences</td>
			<td>555214</td>
			<td>The set contains hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1560</td>
			<td>MirVana RNA isolation kit</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone-based RNA isolation aid</td>
			<td>Life Technologies</td>
			<td>AM9690</td>
			<td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td>
		</tr>
		<tr>
			<td>RNA cleanup kit</td>
			<td>Qiagen Inc.</td>
			<td>74004</td>
			<td>MinElute RNA cleanup kit is designed for purification of up to 45 &#956;g RNA.</td>
		</tr>
		<tr>
			<td>Plasma chamber</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Hitachi Ltd.</td>
			<td>S-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop scanner</td>
			<td>Hewlett-Packard Company</td>
			<td>Photosmart B110</td>
			<td>8-bit color images were captured. Cameras and smart phones may be also used.</td>
		</tr>
		<tr>
			<td>Image-record system</td>
			<td>J&#38;H Technology Co</td>
			<td>GeneSys G:BOX Chemi-XX8</td>
			<td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td>
		</tr>
	</tbody>
</table>

paper-based devices for isolation and characterization of extracellular vesicles

Vesículas extracelulares (SVE), partículas membranoso liberados a partir de vários tipos de células, realizar um grande potencial para aplicações clínicas. Eles contêm ácido nucleico e proteína de carga e são cada vez mais reconhecido como um meio de comunicação intercelular utilizado por ambos eucariota e células procarióticas. No entanto, devido ao seu pequeno tamanho, os protocolos para o isolamento de veículos eléctricos são muitas vezes moroso, complicado e requer grandes volumes de amostra e equipamento caro, tal como uma ultracentrífuga. Para lidar com essas limitações, nós desenvolvemos uma plataforma de imunoafinidade baseado em papel para separar subgrupos de EVs que é fácil, eficiente e requer volumes de amostra tão baixas como 10 jul. As amostras biológicas podem ser pipetados directamente para zonas de teste de papel que tenham sido quimicamente modificados com moléculas de captura que têm uma afinidade elevada para marcadores de superfície específicos EV. Immunosorben ligado a enzima Nós validar o ensaio por meio de microscopia eletrônica de varredura (MEV), com base em papelensaios t (P-ELISA), e análise de transcriptoma. Estes dispositivos baseados em papel permitirá o estudo de EVs na clínica e no ambiente de pesquisa para ajudar a avançar a nossa compreensão das funções EV na saúde e na doença.

A capacidade do dispositivo de papel para isolar subconjuntos de veículos eléctricos assenta de forma eficiente mediante o reconhecimento sensível e específico de marcadores de superfície EV. A modificação das fibras de papel estável com moléculas de captura é conseguido usando a química da avidina-biotina, conforme descrito noutro local 28-30. A eficácia de conjugação química e que o método de fisiossorção é avaliada utilizando leituras baseados em fluorescência. As zonas de teste de pape...

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Este protocolo detalha um método para isolar vesículas extracelulares (SVE), pequenas partículas membranoso liberados a partir de células, a partir de tão pouco como 10 amostras de soro ul. Esta abordagem evita a necessidade de ultracentrifugação, exige apenas alguns minutos de tempo de ensaio, e permite o isolamento de VE a partir de amostras de volumes limitados.

Os dispositivos à base de papel para o Isolamento e Caracterização de Extracelular Vesículas

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

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Research

JoVE Journal

Bioengineering

11.3K visualizações.  National Tsing Hua University.  Este protocolo detalha um método para isolar vesículas extracelulares (SVE), pequenas partículas membranoso liberados a partir de células, a partir de tão pouco como 10 amostras de soro ul. Esta abordagem evita a necessidade de ultracentrifugação, exige apenas alguns minutos de tempo de ensaio, e permite o isolamento de VE a partir de amostras de volumes limitados. 

Vídeo: Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Quantificação e Tamanho-profiling de vesículas extracelulares Usando Sintonizável resistiva Pulso Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Bioengenharia

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabricação de dispositivos microfluídicos tridimensional baseados em papel para imunoensaios

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Avaliação da estabilidade do armazenamento de vesículas extracelulares

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Fabricação rápida de dispositivos microfluídicos personalizados para pesquisa e aplicações educacionais

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

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, ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Rotulagem de vesículas extracelulares para monitoramento de migração e absorção em Explants de cartilagem

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Melhorando a reprodutibilidade para atender informações mínimas para estudos de diretrizes extracelulares de vesículas 2018 em análise de rastreamento de nanopartículas

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

Estudo de Projeto para Implementação para Desenvolvimento e Validação de Pacientes de Diagnósticos de Interruptores de Retenção de Pé Baseados em Papel

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Avaliação da Terapia de Modulação da Contratilidade Cardíaca em Cardiomiócitos Derivados de Células-Tronco Humanas 2D

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...