Papir-baserede enheder til isolering og karakterisering af ekstracellulær Vesikler
Summary
Denne protokol beskriver en metode til at isolere ekstracellulære vesikler (EVS), små hindeagtige partikler frigives fra cellerne, fra så lidt som 10 pi serumprøver. Denne fremgangsmåde omgår behovet for ultracentrifugering, kræver kun nogle få minutters assay tid og muliggør isolering af elbiler fra prøver af begrænsede mængder.
Abstract
Ekstracellulære vesikler (EVS), hindeagtige partikler frigivet fra forskellige typer af celler, holde et stort potentiale for kliniske anvendelser. De indeholder nukleinsyre og protein fragt og er i stigende grad anerkendt som et middel til intercellulær kommunikation anvendes af både eukaryot og prokaryote celler. Men på grund af deres lille størrelse, nuværende protokoller til isolering af EVT ofte tidskrævende, besværligt og kræver store prøvevolumener og dyrt udstyr, såsom en ultracentrifuge. For at løse disse begrænsninger har vi udviklet et papirbaseret immunoaffinitets- platform til adskillelse undergrupper af elbiler, der er let, effektiv og kræver prøvevolumener så lave som 10 pi. Biologiske prøver kan pipetteret direkte på papir testzoner, der er blevet kemisk modificeret med indfangningsmolekyler, der har høj affinitet til specifikke EV overflademarkører. Vi validere assayet ved anvendelse af scanningselektronmikroskopi (SEM), papirbaserede enzymkoblet immunosorbent assays (P-ELISA) og transkriptom analyse. Disse papirbaserede enheder vil gøre det muligt at studere elbiler i klinikken og indstillingen forskning for at hjælpe fremme vores forståelse af EV funktioner i sundhed og sygdom.
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
De mest kritiske trin for en vellykket isolering af undergrupper af ekstracellulære vesikler er: 1) et godt valg af papir; 2) stabil og høj dækning af indfangningsmolekyler på overfladen af papirfibre; 3) korrekt håndtering af prøver; og 4) almen laboratorium hygiejnepraksis.
Porøse materialer er blevet anvendt i mange billige og udstyr-fri assays. De kan have afstemmelige porestørrelse, alsidig funktionalitet, lave omkostninger og høj overflade-til-volumen-forhold tillader pa…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet delvist af Taiwan National Science Rådet grants- NSC 99-2320-B-007-005-MY2 (CC) og NSC 101-2628-E-007-011-MY3 (CMC) og Veterans General Hospitaler og University System af Taiwan fælles forskningsprogram (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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