Dispositivos basados en papel para aislamiento y caracterización de extracelular vesículas
Summary
Este protocolo detalla un método para aislar vesículas extracelulares (SVE), pequeñas partículas membranosas liberadas de las células, a partir de tan poco como 10 muestras de suero l. Este enfoque evita la necesidad de ultracentrifugación, requiere sólo unos pocos minutos de tiempo de ensayo, y permite el aislamiento de los vehículos eléctricos a partir de muestras de volúmenes limitados.
Abstract
Vesículas extracelulares (EVs), partículas membranosas liberados de varios tipos de células, tienen un gran potencial para aplicaciones clínicas. Contienen ácido nucleico y proteínas de carga y se reconoce cada vez más como un medio de comunicación intercelular utilizado tanto por eucariota y células procariotas. Sin embargo, debido a su pequeño tamaño, los protocolos actuales para el aislamiento de los vehículos eléctricos son a menudo consume mucho tiempo, engorroso, y requieren grandes volúmenes de muestras y equipos caros, tales como una ultracentrífuga. Para hacer frente a estas limitaciones, hemos desarrollado una plataforma de inmunoafinidad en papel para separar subgrupos de vehículos eléctricos que es fácil, eficiente y requiere volúmenes de muestra tan bajas como 10 l. Las muestras biológicas pueden ser pipetearon directamente sobre zonas de ensayo de papel que han sido modificados químicamente con moléculas de captura que tienen alta afinidad a los marcadores específicos de la superficie EV. Immunosorben ligado a enzimas Validamos el ensayo mediante el uso de microscopía electrónica de barrido (SEM), en papelensayos t (P-ELISA), y el análisis del transcriptoma. Estos dispositivos basados en papel permitirán el estudio de los vehículos eléctricos en la clínica y el marco de la investigación para ayudar a avanzar nuestra comprensión de las funciones de EV en la salud y la enfermedad.
Introduction
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.
Protocol
Representative Results
Discussion
Los pasos más críticos para el éxito en el aislamiento de los subgrupos de vesículas extracelulares son: 1) una buena opción de papel; 2) la cobertura estable y alto de moléculas de captura en la superficie de las fibras de papel; 3) el manejo adecuado de las muestras; y 4) las prácticas de higiene general de laboratorio.
Los materiales porosos se han utilizado en muchos ensayos baratas y equipo libre. Pueden tener el tamaño de poro sintonizable, funcionalidad versátil, de bajo cost…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Esta obra fue financiada en parte por el Consejo de Seguridad Nacional 99-2320-B-007-005-MY2 el Consejo Nacional de Ciencia de Taiwán grants- (CC) y NSC 101-2628-E-007-011-MY3 (CMC), y el General de Veteranos Hospitales y Sistema Universitario del Programa Común de Investigación de Taiwán (CC).
Materials
| Chromatography Paper | GE Healthcare Life Sciences | 3001-861 | Whatman® Grade 1 cellulose paper |
| (3-Mercaptopropyl) trimethoxysilane | Sigma Aldrich | 175617 | 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. |
| Ethanol | Fisher Scientific | BP2818 | Absolute, 200 Proof, molecular biology grade |
| Bovine serum albumin (BSA) | BioShop Canada Inc. | ALB001 | Often referred to as Cohn fraction V. |
| N-g-maleimidobutyryloxy succinimide ester (GMBS) | Pierce Biotechnology | 22309 | GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive. |
| Avidin | Pierce Biotechnology | 31000 | NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin |
| Biotinylated mouse anti-human anti-CD63 | Ancell | 215-030 | clone AHN16.1/46-4-5 |
| biotinylated annexin V | BD Biosciences | 556418 | Annxin V has a high affinity for phosphotidylserine (PS) |
| Primary anti-CD9 and secondary antibody | System Biosciences | EXOAB-CD9A-1 | The secondary antibody is horseradish peroxidise-conjugated |
| Serum separation tubes | BD Biosciences | 367991 | Clot activator and gel for serum separation |
| Annexin V binding buffer | BD Biosciences | 556454 | 10X; dilute to 1X prior to use. |
| TMB substrate reagent set | BD Biosciences | 555214 | The set contains hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB) |
| RNA isolation kit | Life Technologies | AM1560 | MirVana RNA isolation kit |
| Polyvinylpyrrolidone-based RNA isolation aid | Life Technologies | AM9690 | Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides. |
| RNA cleanup kit | Qiagen Inc. | 74004 | MinElute RNA cleanup kit is designed for purification of up to 45 μg RNA. |
| Plasma chamber | March Instruments | PX-250 | |
| Scanning electron microscope | Hitachi Ltd. | S-4300 | |
| Desktop scanner | Hewlett-Packard Company | Photosmart B110 | 8-bit color images were captured. Cameras and smart phones may be also used. |
| Image-record system | J&H Technology Co | GeneSys G:BOX Chemi-XX8 | 16-bit fluroscence images were captured. Fluroscence microscopes may be also used. |
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