Optagelse af fluorescerende mærket små ekstracellulære vesikler In Vitro og i rygmarven
Summary
Vi beskriver en protokol til mærkning makrofag-afledte små ekstracellulære vesikler med PKH farvestoffer og observere deres optagelse in vitro og i rygmarven efter intrathecal levering.
Abstract
Små ekstracellulære vesikler (SEVs) er 50-150 nm vesikler udskilles af alle celler og til stede i kropsvæsker. sEVs overføre biomolekyler såsom RNA, proteiner og lipider fra donor til acceptor celler, hvilket gør dem centrale signalering mæglere mellem celler. I centralnervesystemet (CNS), sEVs kan mægle intercellulære signalering, herunder neuroimmune interaktioner. sEV-funktioner kan studeres ved at spore optagelsen af mærkede SEVs i modtagerceller både in vitro og in vivo. Dette papir beskriver mærkning af sEVs fra de betingede medier af RAW 264.7 makrofag celler ved hjælp af en PKH membran farvestof. Det viser udbredelsen af forskellige koncentrationer af mærket sEVs på flere tidspunkter af Neuro-2a celler og primære astrocytter in vitro. Også vist er optagelse af SEVs leveret intrathecally i musen rygmarvs neuroner, astrocytter, og mikroglia visualiseret af konfokal mikroskopi. De repræsentative resultater viser tidsafhængig variation i optagelsen af sEVs af forskellige celler, som kan hjælpe med at bekræfte vellykket SEVs levering i rygmarven.
Introduction
Små ekstracellulære vesikler (SEVs) er nanosized, membran-afledte vesikler med en størrelse på 50-150 nm. De stammer fra multi-vesikulære organer (MVB) og frigives fra celler ved fusion af MVB’erne med plasmamembranen. sEVs indeholder miRNA’er, mRNAs, proteiner og bioaktive lipider, og disse molekyler overføres mellem celler i form af celle-til-celle kommunikation. sEVs kan internaliseres af modtagerceller ved hjælp af en række endoytiske veje, og denne fangst af sEVs af modtagerceller medieres ved genkendelse af overflademolekyler på både elbiler og målcellerne1.
SEVs har fået interesse på grund af deres evne til at udløse molekylære og fænotypiske ændringer i acceptorceller, deres nytte som terapeutisk middel, og deres potentiale som bærere for last molekyler eller farmakologiske agenser. På grund af deres lille størrelse kan billeddannelse og sporing af SEVs være udfordrende, især for in vivo-undersøgelser og kliniske indstillinger. Derfor er der udviklet mange metoder til at mærke og image sEVs til at hjælpe deres biodistribution og sporing in vitro og in vivo2.
Den mest almindelige teknik til at studere sEV biodistribution og målcelleinteraktioner indebærer mærkning af dem med fluorescerende farvemolekyler3,4,5,6,7. Elbiler blev oprindeligt mærket med cellemembranfarvestoffer, der almindeligvis blev brugt til billedceller. Disse fluorescerende farvestoffer generelt plette lipid bilayer eller proteiner af interesse på SEVs. Flere lipofile farvestoffer viser et stærkt fluorescerende signal, når de indgår i cytosolen, herunder DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine jod), DiL (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indocarbocyanine perchlorat), og DiD (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indocarbocyanine 4-chlorobenzenesulfonat salt)8,9,10,11.
Andre lipofile farvestoffer, såsom PKH67 og PKH26, har en meget fluorescerende polar hovedgruppe og en lang alifatisk kulbrintehale, der let interkalker i enhver lipidstruktur og fører til langsigtet farvefastholdelse og stabil fluorescens12. PKH farvestoffer kan også mærke elbiler, som gør det muligt at studere EV egenskaber in vivo13. Mange andre farvestoffer er blevet brugt til at observere exosomer ved hjælp af fluorescensmikroskopi og flowcytometri, herunder lipidmærkningsfarvestoffer14 og cellepermeable farvestoffer som carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)15,16 og calcein acetoxymethyl (AM) ester17.
Undersøgelser af sEV-medieret krydstale mellem forskellige celler i CNS har givet vigtig indsigt i patogenesen af neuroinflammatoriske og neurodegenerative sygdomme18. For eksempel kan SEVs fra neuroner sprede beta-amyloid peptider og fosforylerede tau proteiner og støtte i patogenese af Alzheimers sygdom19. Derudover indeholder elbiler afledt af erythrocytter store mængder alfa-synuclein og kan krydse blod – hjerne barrieren og bidrage til Parkinsons patologi20. SEVs evne til at krydse fysiologiske barrierer21 og overføre deres biomolekyler til at målrette celler gør dem praktiske værktøjer til at levere terapeutiske lægemidler til CNS22.
Visualisering af SEV optagelse af utallige CNS celler i rygmarven vil muliggøre både mekanistiske undersøgelser og evaluering af de terapeutiske fordele ved eksogent administrerede sEVs fra forskellige cellulære kilder. Dette papir beskriver metoden til at mærke sEVs stammer fra makrofager og billede deres optagelse in vitro og in vivo i lændemarven af neuroner, mikroglia, og astrocytter til kvalitativt bekræfte sEV levering ved visualisering.
Protocol
Representative Results
Discussion
I denne protokol viste vi mærkning af SEVs med PKH farvestoffer og visualisering af deres optagelse i rygmarven. PKH lipofile fluorescerende farvestoffer anvendes i vid udstrækning til mærkning af celler efter flowcytometri og fluorescerende mikroskopi3,5,6,12,24,25. På grund af deres relativt lange halveringstid og lave …
Declarações
The authors have nothing to disclose.
Acknowledgements
Denne undersøgelse blev støttet af tilskud fra NIH NINDS R01NS102836 og Pennsylvania Department of Health Commonwealth Universal Research Enhancement (CURE) tildelt Seena K. Ajit. Vi takker Dr. Bradley Nash for kritisk læsning af manuskriptet.
Materials
| Amicon Ultra 0.5 mL centrifugal filters | MilliporeSigma | Z677094 | |
| Anti-Alix Antibody | Abcam | ab186429 | 1:1000 |
| Anti-Calnexin Antibody | Abcam | Ab10286 | 1:1000 |
| Anti-CD81 Antibody | Santa Cruz Biotechnology | sc-166029 | 1:1000 |
| Anti-GAPDH Monoclonal Antibody (14C10) | Cell Signaling Technology | 2118 | 1:1000 |
| Anti-Glial Fibrillary Acidic Protein Antibody | Sigma-Aldrich | MAB360 | 1:500 for IF; 1:1000 for IHC |
| Anti-Iba1 Antibody | Wako | 019-19741 | 1:2000 |
| Anti-MAP2A Antibody | Sigma-Aldrich | MAB378 | 1:500 |
| Bovine Serum Albumin (BSA) | VWR | 0332 | |
| Cell Strainer, 40 μm | VWR | 15-1040-1 | |
| Centrifuge Tubes | Thermo Scientific | 3118-0050 | 12,000 x g |
| Coverslip, 12-mm, #1.5 | Electron Microscopy Sciences | 72230-01 | |
| Coverslip, 18-mm, #1.5 | Electron Microscopy Sciences | 72222-01 | |
| DAPI | Sigma-Aldrich | D9542-1MG | 1 µg/mL |
| DC Protein Assay | Bio-Rad | 500-0116 | |
| Deoxyribonuclease I (DNAse I) | MilliporeSigma | D4513-1VL | |
| Donkey Anti-Rabbit IgG H&L (HRP) | Abcam | ab16284 | 1:10000 |
| Donkey Anti-Rabbit IgG H&L, Alexa Fluor 488 | Invitrogen | A-21206 | 1:500 |
| Double Frosted Microscope Slides, #1 | Thermo Scientific | 12-552-5 | |
| DPBS without Calcium and Magnesium | Corning | 21-031-CV | |
| Dulbecco's Modified Eagle Medium (DMEM) | Corning | 10-013-CV | |
| Exosome-Depleted Fetal Bovine Serum | Gibco | A27208-01 | |
| Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
| FluorChem M imaging system | ProteinSimple | ||
| FV3000 Confocal Microscope | Olympus | ||
| Goat Anti-Mouse IgG H&L (HRP) | Abcam | ab6789 | 1:10000 |
| Goat Anti-Mouse IgG H&L, Alexa Fluor 488 | Invitrogen | A-11001 | 1:500 |
| Goat Anti-Mouse IgG1, Alexa Fluor 594 | Invitrogen | A-21125 | |
| Hank's Balanced Salt Solution (HBSS) | VWR | 02-0121 | |
| HEPES | Gibco | 15630080 | |
| HRP Substrate | Thermo Scientific | 34094 | |
| Intercept blocking buffer, TBS | LI-COR Biosciences | 927-60001 | |
| Laemmli SDS Sample Buffer | Alfa Aesar | AAJ61337AC | |
| Micro Cover Glass, #1 | VWR | 48404-454 | |
| Microm HM550 | Thermo Scientific | ||
| NanoSight NS300 system | Malvern Panalytical | ||
| NanoSight NTA 3.2 software | Malvern Panalytical | ||
| Neuro-2a Cell Line | ATCC | CCL-131 | |
| Normal Goat Serum | Vector Laboratories | S-1000 | |
| O.C.T Compound | Sakura Finetek | 4583 | |
| Papain | Worthington Biochemical Corporation | NC9597281 | |
| Paraformaldehyde | Electron Microscopy Sciences | 19210 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| PKH26 | Sigma-Aldrich | MINI26-1KT | |
| PKH67 | Sigma-Aldrich | MINI67-1KT | |
| Protease Inhibitor Cocktail | Thermo Scientific | 1862209 | |
| PVDF Transfer Membrane | MDI | SVFX8302XXXX101 | |
| RAW 267.4 Cell Line | ATCC | TIB-71 | |
| RIPA Buffer | Sigma-Aldrich | R0278 | |
| Sodium Chloride | AMRESCO | 0241-2.5KG | |
| Superfrost Plus Gold Slides | Thermo Scientific | 15-188-48 | adhesive slides |
| T-75 Flasks | Corning | 431464U | |
| Tecnai 12 Digital Transmission Electron Microscope | FEI Company | ||
| TEM Grids | Electron Microscopy Sciences | FSF300-cu | |
| Tris-Glycine Protein Gel, 12% | Invitrogen | XP00120BOX | |
| Tris-Glycine SDS Running Buffer | Invitrogen | LC26755 | |
| Tris-Glycine Transfer Buffer | Invitrogen | LC3675 | |
| TrypLE Express | cell dissociation enzyme | ||
| Triton X-100 | Acros Organics | 327371000 | |
| Trypsin, 0.25% | Corning | 25-053-CL | |
| Tween 20 | |||
| Ultracentrifuge Tubes | Beckman | 344058 | 110,000 x g |
Referências
- Mulcahy, L. A., Pink, R. C., Carter, D. R. F. Routes and mechanisms of extracellular vesicle uptake. Journal of Extracellular Vesicles. 3 (1), 24641 (2014).
- Betzer, O., et al. Advances in imaging strategies for in vivo tracking of exosomes. Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. 12 (2), 1594 (2020).
- Dehghani, M., Gaborski, T. R. Fluorescent labeling of extracellular vesicles. Methods in Enzymology. 645, 15-42 (2020).
- González, M. I., et al. Covalently labeled fluorescent exosomes for in vitro and in vivo applications. Biomedicines. 9 (1), 81 (2021).
- Chuo, S. T. -. Y., Chien, J. C. -. Y., Lai, C. P. -. K. Imaging extracellular vesicles: current and emerging methods. Journal of Biomedical Science. 25 (1), 91 (2018).
- vander Vlist, E. J., Nolte-‘tHoen, E. N. M., Stoorvogel, W., Arkesteijn, G. J. A., Wauben, M. H. M. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nature Protocols. 7 (7), 1311-1326 (2012).
- Hoshino, A., et al. Tumour exosome integrins determine organotropic metastasis. Nature. 527 (7578), 329-335 (2015).
- Heinrich, L., et al. Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials. Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 81 (1), 153-161 (2007).
- Haney, M. J., et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. Journal of Controlled Release. 207, 18-30 (2015).
- Grange, C., et al. Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging. International Journal of Molecular Medicine. 33 (5), 1055-1063 (2014).
- Wiklander, O. P., et al. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. Journal of Extracellular Vesicles. 4, 26316 (2015).
- Lai, C. P., et al. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters. Nature Communications. 6 (1), 7029 (2015).
- Deddens, J. C., et al. Circulating extracellular vesicles contain miRNAs and are released as early biomarkers for cardiac injury. Journal of Cardiovascular Translational Research. 9 (4), 291-301 (2016).
- Montecalvo, A., et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood. 119 (3), 756-766 (2012).
- Escrevente, C., Keller, S., Altevogt, P., Costa, J. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer. 11, 108 (2011).
- Cho, E., et al. Comparison of exosomes and ferritin protein nanocages for the delivery of membrane protein therapeutics. Journal of Controlled Release. 279, 326-335 (2018).
- Mantel, P. Y., et al. Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system. Cell Host & Microbe. 13 (5), 521-534 (2013).
- Porro, C., Trotta, T., Panaro, M. A. Microvesicles in the brain: Biomarker, messenger or mediator. Journal of Neuroimmunology. 288, 70-78 (2015).
- De Toro, J., Herschlik, L., Waldner, C., Mongini, C. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Frontiers in Immunology. 6, 203 (2015).
- Matsumoto, J., et al. Transmission of alpha-synuclein-containing erythrocyte-derived extracellular vesicles across the blood-brain barrier via adsorptive mediated transcytosis: another mechanism for initiation and progression of Parkinson’s disease. Acta Neuropathologica Communications. 5 (1), 71 (2017).
- Matsumoto, J., Stewart, T., Banks, W. A., Zhang, J. The transport mechanism of extracellular vesicles at the blood-brain barrier. Current Pharmaceutical Design. 23 (40), 6206-6214 (2017).
- Shaimardanova, A., et al. Extracellular vesicles in the diagnosis and treatment of central nervous system diseases. Neural Regeneration Research. 15 (4), 586-596 (2020).
- Théry, C., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).
- Hoen, E. N. M. N. -. t., et al. Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine: Nanotechnology, Biology and Medicine. 8 (5), 712-720 (2012).
- Gangadaran, P., Hong, C. M., Ahn, B. -. C. An update on in vivo imaging of extracellular vesicles as drug delivery vehicles. Frontiers in Pharmacology. 9, 169 (2018).
- Teare, G. F., Horan, P. K., Slezak, S. E., Smith, C., Hay, J. B. Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes. Cellular Immunology. 134 (1), 157-170 (1991).
- Kuffler, D. P. Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs. Journal of Comparative Neurology. 302 (4), 729-738 (1990).
- Takov, K., Yellon, D. M., Davidson, S. M. Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes. Journal of Extracellular Vesicles. 6 (1), 1388731 (2017).
- Pužar Dominkuš, P., et al. PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles. Biochimica et Biophysica Acta. Biomembranes. 1860 (6), 1350-1361 (2018).
- Dehghani, M., Gulvin, S. M., Flax, J., Gaborski, T. R. Systematic evaluation of PKH labelling on extracellular vesicle size by nanoparticle tracking analysis. Scientific Reports. 10 (1), 9533 (2020).
- Shimomura, T., et al. New lipophilic fluorescent dyes for labeling extracellular vesicles: characterization and monitoring of cellular uptake. Bioconjugate Chemistry. 32 (4), 680-684 (2021).
- Yuan, D., et al. Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain. Biomaterials. 142, 1-12 (2017).
- Polanco, J. C., Li, C., Durisic, N., Sullivan, R., Götz, J. Exosomes taken up by neurons hijack the endosomal pathway to spread to interconnected neurons. Acta Neuropathologica Communications. 6 (1), 10 (2018).
- Jurgielewicz, B. J., Yao, Y., Stice, S. L. Kinetics and specificity of HEK293T extracellular vesicle uptake using imaging flow cytometry. Nanoscale Research Letters. 15 (1), 170 (2020).
