Dispositifs à base de papier pour l'isolement et la caractérisation de vésicules extracellulaire
Summary
Ce protocole décrit en détail une méthode pour isoler des vésicules extracellulaires (VE), de petites particules libérées par des cellules membranaires, d'aussi peu que 10 échantillons de sérum ul. Cette approche évite la nécessité d'ultracentrifugation, ne nécessite que quelques minutes de temps de dosage, et permet l'isolement des véhicules électriques à partir d'échantillons de volumes limités.
Abstract
Vésicules extracellulaires (VE), membraneuses particules libérées par divers types de cellules, détiennent un grand potentiel pour des applications cliniques. Ils contiennent cargaison d'acide nucléique et des protéines et sont de plus en plus reconnus comme un moyen de communication intercellulaire utilisé à la fois par eucaryote et les cellules procaryotes. Toutefois, en raison de leur petite taille, les protocoles actuels pour l'isolement des véhicules électriques sont souvent beaucoup de temps, lourde, et nécessitent de grands volumes d'échantillons et des équipements coûteux, comme une ultracentrifugation. Pour répondre à ces limitations, nous avons développé une plate-forme d'immunoaffinité à base de papier pour séparer les sous-groupes de véhicules électriques qui est facile, efficace et nécessite des volumes d'échantillon aussi faibles que 10 pi. Les échantillons biologiques peuvent être introduits à la pipette directement sur les zones de test de papier qui ont été chimiquement modifiés avec des molécules de capture ayant une affinité élevée à des marqueurs spécifiques de la surface d'exposition. Immunosorben lié à une enzyme, à base de papier Nous valider l'essai en utilisant la microscopie électronique à balayage (MEB)t essais (P-ELISA), et l'analyse du transcriptome. Ces dispositifs à base de papier permettront l'étude des véhicules électriques dans la clinique et le contexte de la recherche pour faire avancer notre compréhension des fonctions EV en matière de santé et de la maladie.
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
Les étapes les plus critiques pour l'isolement réussi de sous-groupes de vésicules extracellulaires sont: 1) un bon choix de papier; 2) une couverture stable et élevé de molécules de capture sur la surface des fibres de papier; 3) la manipulation correcte des échantillons; et 4) la pratique de l'hygiène générale de laboratoire.
Les matériaux poreux ont été utilisés dans de nombreux tests peu coûteux et sans équipement. Ils peuvent avoir une taille de pores accordable,…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Ce travail a été financé en partie par NSC 99-2320-B-007-005-MY2 du Conseil national des sciences de Taiwan (CC) et NSC 101-2628-E-007-011-MY3 (CMC) et le Veterans General Hôpitaux et University System of Programme conjoint de recherche de Taiwan (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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