Detta protokoll detaljer en metod för att isolera cellulära vesiklar (EVT), små membranpartiklar som frigörs från celler, från så lite som 10 | il serumprov. Detta tillvägagångssätt kringgår behovet av ultracentrifuge, kräver bara några minuter av analystiden, och möjliggör isolering av elbilar från prover av begränsade volymer.
Cellulära vesiklar (EVS), membranpartiklar som frigörs från olika typer av celler, hålla en stor potential för kliniska tillämpningar. De innehåller nukleinsyra och protein last och erkänns alltmer som ett medel för kommunikationen mellan utnyttjas av både eukaryota och prokaryota celler. Men på grund av sin ringa storlek, nuvarande protokoll för isolering av elbilar är ofta tidskrävande, besvärlig, och kräver stora volymer prov och dyr utrustning, till exempel en ultracentrifug. För att lösa dessa begränsningar, utvecklade vi en pappersbaserad immunoaffinitets plattform för att separera undergrupper av elbilar som är enkelt, effektivt och kräver provvolymer så låga som 10 pl. Biologiska prover kan pipetteras direkt på papperstestzoner som har modifierats kemiskt med infångningsmolekyler som har hög affinitet till specifika EV ytmarkörer. Vi validera analysen genom användning av svepelektronmikroskop (SEM), pappersbaserad enzymkopplad immunosorbent analyser (P-ELISA) och transkriptomanalys. Dessa pappersbaserade enheter kommer att möjliggöra studier av elbilar i kliniken och inställningen forskning för att hjälpa öka vår förståelse av EV funktioner i hälsa och sjukdom.
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.
De mest kritiska stegen för framgångsrik isolering av subgrupper av extracellulära blåsor är: 1) ett bra val av papper; 2) stabil och hög täckning av infångningsmolekyler på ytan av pappersfibrer; 3) korrekt hantering av prover; och 4) allmän laboratoriehygien.
Porösa material har utnyttjats i många billiga och utrustningsfria analyser. De kan ha avstämbar porstorlek, mångsidig funktionalitet, låg kostnad och hög yta-till-volymförhållande medger passiv uppsugning av fluider…
The authors have nothing to disclose.
Detta arbete stöddes delvis av Taiwan National Science Council grants- NSC 99-2320-B-007-005-MY2 (CC) och NSC 101-2628-E-007-011-My3 (CMC), och Veterans General Sjukhus och universitet System of Taiwan Joint Research Program (CC).
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. |