Diese Arbeit wurde zum Teil durch die National Science Council Taiwan-Beihilfen NSC 99-2320-B-007-005-MY2 (CC) und NSC 101-2628-E-007-011-MY3 (CMC), und der Veterans General unterstützt Krankenhäuser und Universitätssystem von Taiwan Joint Research Programme (CC).

Ein allgemeines Diagramm des Betriebs ist in Abbildung 1 zur Verfügung gestellt. Mit ethischen Praktiken, sammelten wir Blutproben von gesunden Probanden und erhalten Kammerwasser-Proben von Patienten durch die Taichung Veterans General Hospital (TCVGH), Taichung, Taiwan unter IRB zugelassen Protokolle ( IRB TCVGH Nr CF11213-1). 1. Herstellung von Papier Material <ol><li> Geschnitten Chromatographiepapier in Kreise von 5 mm im Durchmesser, um das gleiche Layout wie einer 96-Well-Mikrotiterplatte ist. Sandwich diese Papierstücke mit zwei Polystyrolplatten mit einem Stammdurchgangslöchern, und Laminat. </li><li> Ändern Papier Geräte mit dem in den folgenden Schritten 28 beschrieben chemische Konjugation Verfahren. <ol><li> Saures Papier Geräte mit Sauerstoffplasma (100 mW, 1% Sauerstoff, 30 sec) in einer Plasmakammer. ACHTUNG: Besondere Vorsicht ist bei der Arbeit mit 3-Mercaptopropyltrimethoxysilan und N -γ-Maleimidobutyryloxy Succinimid genommen werden(GMBS), da sie sowohl feuchtigkeitsempfindlich und toxisch sind. Schutzhandschuhe und ein Einatmen zu vermeiden oder mit Haut und Augen zu kontaktieren. Halten Sie die Vorratsflasche fest verschlossen und lassen Sie ihn auf Raumtemperatur vor dem Öffnen um eine Kondenswasserbildung zu vermeiden Gleichgewicht. Alternativ öffnen Sie die Vorratsflasche in einem mit Stickstoff gefüllten Handschuhbeutel oder Kasten. Aliquot in kleine Fläschchen häufiges Öffnen der Vorratsflasche zu vermeiden. </li><li> Unmittelbar inkubieren behandelte Papier Vorrichtungen in einer 4% igen (v / v) Lösung von 3-Mercaptopropyltrimethoxysilan in Ethanol (200 proof) für 30 min. </li><li> Papier Geräte Spülen mit Ethanol und Inkubation sie mit 0,01 mmol / ml GMBS in Ethanol für 15 min. </li><li> Spülen mit Ethanol (200 proof) und inkubieren Papier Vorrichtungen mit 10 ug / ml Avidin-Lösung in phosphatgepufferter Salzlösung (PBS) für 1 h bei 4 ° C. Lagerung bei 4 ° C, wenn nötig, und innerhalb von 4 Wochen. </li></ol></li><li> Befeuchten jeder Papiertestzonen mit 10 ul PBS, enthaltend 1% (w / v) Rinderserumalbumin (BSA) Für 3 x 10 min vor Spek 10 ul biotinylierten Fängermoleküle für 3 x 10 min. Verwenden anti CD63-Antikörper (20 ug / ml in PBS, das 1% (w / v) BSA und 0,09% (w / v) Natriumazid) oder Annexin V (1:20, v / v in Annexin V-Bindungspuffer), wie Fängermoleküle. </li><li> Abspülen ungebundenen anti-CD63-Antikörper oder Annexin V-Moleküle unter Verwendung von 10 &amp; mgr; l entspricht, PBS, das 1% (w / v) BSA oder Annexin V-Bindungspuffer Lösungen für 3 × 1 min auf. </li></ol> 2. Serum und Kammerwasser Probenerhebung und Datenverarbeitung <ol><li> Serumgewinnung. <ol><li> Sammeln Sie 10 ml peripheren Blut durch Venenpunktion in Serumtrennröhrchen und sanft das Röhrchen fünfmal. Stellen Sie die Röhre in einer vertikalen Position und für weitere 30 min. </li><li> Zentrifuge bei 1200 × g für 15 min. Das Serum von der oberen Schicht in ein sauberes Röhrchen, und erneut zentrifugieren bei 3.000 × g für 30 min. </li><li> Übergeben Sie den Überstand durch ein 0,8 um filter. Halten Sie die Probe bei -80 ° C bis zur Verwendung. </li></ol></li><li> Kammerwasser Kollektion: sammeln Kammerwasser-Proben von Patienten durch einen Augenarzt direkt durch invasive Verfahren und Lagerung von Proben bei -80 ° C bis zur Verwendung diagnostiziert. </li></ol> 3. Isolierung der extrazellulären Vesikeln <ol><li> Stichproben auf jeder Papiertestzone mit einer Geschwindigkeit von 5 ul / min. </li><li> Abspülen ungebundenen EVs mit 10 ul entsprechenden PBS, das 1% (w / v) BSA oder Annexin V-Bindungspuffer Lösungen für 3 × 1 min für die Papiervorrichtungen funktionalisiert mit Anti-CD63-Antikörper oder Annexin V-Moleküle sind. Führen Sie die folgenden Downstream-Assays. </li></ol> 4. Downstream Testbeispiel 1: Scanning Electromicrographs <ol><li> Fix EVs auf funktionalisierten Papiertestzone mit 10 ul 0,5 x Karnovsky Fixativ für 10 Minuten eingefangen. </li><li> Spülen Sie die Proben mit ausreichend PBS für 2 x 5 min. </li><li> Entwässern derProben mit anschließender 35% Ethanol für 10 min, 50% Ethanol für 2 × 10 min, 70% Ethanol für 2 x 10 min, 95% Ethanol für 2 × 10 min und 100% Ethanol für 4 x 10 min. </li><li> Critical trocken und Sputter-Beschichtung der Proben mit Palladium / Gold und untersuchen mit einem Rasterelektronenmikroskop bei geringer Elektronenbeschleunigungsspannung betrieben (~ 5 kV). </li></ol> 5. Downstream Testbeispiel 2: Papier-basierten ELISA <ol><li> Fügen Sie ein 5 ul-Lösung, die Anti-CD9 primären Antikörper bei 1: 1000 Verdünnung in PBS zu jeder Testzone, die gefangen genommen EVs. Warten Sie 1 Minute. </li><li> Mit 30 Spülen Sie die Proben ml PBS bei 100 Umdrehungen pro Minute für 30 Sekunden geschüttelt. </li><li> Hinzufügen einer 5 &amp; mgr; l Lösung, die Meerrettich-Peroxidase (HRP) -verknüpften-sekundären Antikörper bei 1: 1000 Verdünnung in PBS und 1 min warten. </li><li> Mit 30 Spülen Sie die Proben ml PBS bei 100 Umdrehungen pro Minute für 30 Sekunden geschüttelt. </li><li> Hinzufügen eines kolorimetrischen Substrats 5 ul, das 1: 1-Volumenverhältnis von Wasserstoffperoxid eind 3,3 &#39;, 5,5&#39;-Tetramethylbenzidin (TMB) und scannen Sie mit einem Desktop-Scanner. </li></ol> 6. Nachgeschaltete Testbeispiel 3: RNA-Isolierung <ol><li> Tauchen Sie die Proben in 35 ul von Polyvinylpyrrolidon-basierte RNA-Isolierung Hilfe und 265 ul Lyse / Bindungspuffer auf EVs erfasst lysieren. Vortex kräftig. </li><li> In 30 ul Homogenat bei der Isolation Kit zum Lysat zur Verfügung gestellt. Vortex kräftig und auf Eis für 10 Minuten gestellt. </li><li> Extrahieren der RNA unter Verwendung von Säure in den folgenden Schritten beschrieben, mit Phenol-Chloroform-Trennung. <ol><li> Nehmen 330 ul Phenol-Chloroform von der Bodenschicht der Flasche und in den Homogenats / Lysat. Vortex kräftig. </li><li> Zentrifugieren bei 10000 × g für 5 min. Übertragen Sie die wässrige (obere) Phase in ein sauberes Röhrchen und das Volumen entfernt. </li></ol></li><li> Fällt die Gesamt-RNA mit 100% Ethanol das Volumen, das 1,25-fache des Volumens der wäßrigen Phase in der vorhergehenden Stufe und co entfernt istllect die RNA in der Elutionslösung auf 95 ° C vorgewärmt. </li><li> Eine Konzentration und weiteren Reinigung der RNA unter Verwendung eines RNA-Cleanup Kit nach dem Protokoll des Herstellers. </li></ol>

<ol>
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Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

Die wichtigsten Schritte zur erfolgreichen Isolierung von Untergruppen von extrazellulären Vesikeln sind: 1) eine gute Wahl des Papiers; 2) stabile und hohe Abdeckung der Fängermoleküle auf der Oberfläche der Papierfasern; 3) die richtige Handhabung der Proben; und 4) allgemeine Laborhygienemaßnahmen. Poröse Materialien haben sich in vielen preiswerten und Ausrüstung freien Tests genutzt. Sie haben abstimmbare Porengrße, vielseitige Funktionalität, niedrige Kosten und hohe Oberfläc...

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.

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	<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

Extrazellulären Vesikeln (EVs), membranartige Partikel aus verschiedenen Arten von Zellen freigesetzt, halten ein großes Potenzial für die klinische Anwendung. Sie enthalten von Nukleinsäuren und Proteinen Ladung und werden zunehmend als Mittel zur interzellulären Kommunikation sowohl eukaryote und prokaryote Zellen verwendet erfasst. Aufgrund ihrer geringen Größe, aktuelle Protokolle zur Isolierung von Elektrofahrzeugen sind oft zeitaufwendig, umständlich und erfordert große Probenvolumina und teure Ausrüstung, wie zum Beispiel einer Ultrazentrifuge. Um diese Einschränkungen zu begegnen, haben wir eine papierbasierte Immunaffinitäts Plattform zur Trennung von Untergruppen von Elektrofahrzeugen, die einfach, effizient ist und erfordert Probenvolumen von nur 10 &amp; mgr; l. Biologische Proben können direkt auf Papier Testzonen, die chemisch mit Fängermolekülen, die eine hohe Affinität zu bestimmten EV Oberflächenmarker haben die modifiziert wurden, pipettiert werden. Wir bestätigen die Assays unter Verwendung von Rasterelektronenmikroskopie (SEM), auf Papierbasis enzymgebundenen immunosorbent-Tests (P-ELISA) und Transkriptom-Analyse. Diese Papier-basierte Geräte ermöglichen das Studium von Elektrofahrzeugen in der Klinik und der Forschung Einstellung zu helfen unser Verständnis von EV-Funktionen in Gesundheit und Krankheit.

Die Fähigkeit des Papiers Gerät Gruppen von EVs isolieren stützt effizient auf seine empfindliche und spezifische Erkennung von EV-Oberflächenmarker. Die stabile Modifikation von Papierfasern mit Fängermolekülen wird durch die Verwendung von Avidin-Biotin-Chemie als anderswo 28-30 beschrieben ist. Die Wirksamkeit der chemischen Konjugation, und dass der Physisorption Verfahren unter Anwendung der fluoreszenzbasierten Auslesungen beurteilt. Die Papier Prüfzonen folgenden Protokoll Schritt 1) ​​mit A...

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

Diese Protokollinformationen ein Verfahren zur extrazellulären Vesikeln (EVs), kleine membranöse Teilchen aus Zellen freigesetzt zu isolieren, von so wenig wie 10 ul Serumproben. Dieser Ansatz umgeht die Notwendigkeit von Ultrazentrifugation, nur wenige Minuten von Testzeit erfordert und ermöglicht die Isolation von Elektrofahrzeugen aus Proben von begrenzten Mengen.

Auf Papier basierende Vorrichtungen zur Isolierung und Charakterisierung von extrazellulärer Vesikel

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

paper-based-devices-for-isolation-characterization-extracellular

Research

JoVE Journal

Bioengineering

11.3K Aufrufe.  National Tsing Hua University.  Diese Protokollinformationen ein Verfahren zur extrazellulären Vesikeln (EVs), kleine membranöse Teilchen aus Zellen freigesetzt zu isolieren, von so wenig wie 10 ul Serumproben. Dieser Ansatz umgeht die Notwendigkeit von Ultrazentrifugation, nur wenige Minuten von Testzeit erfordert und ermöglicht die Isolation von Elektrofahrzeugen aus Proben von begrenzten Mengen. 

Serie: 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.

Quantifizierung und Größe-Profilierung der extrazellulären Vesikeln Mit Tunable Resistive Pulse Sensing

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

Forschung

Biotechnik

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.

Herstellung von dreidimensionalen Papierbasis Microfluidic Devices für Immunoassays

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.

Nachweis von Endotoxin im Nano-Rezepturen mit Limulus Amoebocyte Lysate (LAL) Assays

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.

Bewertung der Speicherstabilität von extrakellulären Bläschen

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.

Schnelle Herstellung von kundenspezifischen mikrofluidischen Geräten für Forschungs- und Bildungsanwendungen

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.

Elektrische und magnetische Feldgeräte zur Stimulation biologischer Gewebe

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.

Markierung extrazellulärer Vesikel zur Überwachung der Migration und Aufnahme in Knorpelexplantaten

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.

Verbesserung der Reproduzierbarkeit, um minimale Informationen für Studien zu extrazellulären Vesikeln zu erfüllen Richtlinien 2018 in der Nanopartikel-Tracking-Analyse

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.

Design-to-Implementierungsstudie zur Entwicklung und Patientenvalidierung der papierbasierten Zehenschalterdiagnostik

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.

Evaluierung der kardialen Kontraktilitätsmodulationstherapie in 2D-Kardiomyozyten aus humanen Stammzellen

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