Name: isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells
Uploaded: 2022-09-23T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells at JoVE.com

Dette arbejde blev støttet af tilskud fra National Natural Science Foundation of China (32000974, 81930025 og 82170988) og China Postdoctoral Science Foundation (2019M663986 og BX20190380). Vi er taknemmelige for hjælpen fra National Experimental Teaching Demonstration Center for Basic Medicine (AMFU) og Analytical and Testing Central Laboratory of Military Medical Innovation Center of Air Force Medical University.

Alle dyreforsøg blev godkendt af Animal Care and Use Committee of the Fourth Military Medical University og udført i overensstemmelse med National Institutes of Health Guide for Care and Use of Laboratory Animals. Otte uger gamle C57Bl/6 mus (ingen præference for hverken hunner eller hanner) blev brugt. Kryopræserverede MSC'er afledt af human navlestreng (UCMSC'er), der blev anvendt til denne undersøgelse, blev indhentet fra en kommerciel kilde (se materialetabel). Anvendelsen af humane celler blev ...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+isolation+of+extracellular+vesicles+by+size-exclusion+chromatography.">Single-step isolation of extracellular vesicles by size-exclusion chromatography.</a> Journal of Extracellular Vesicles. 3, (2014).</li><li>Chen, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteins+in+extracellular+vesicles+as+candidate+markers+for+breast+cancer.">Phosphoproteins in extracellular vesicles as candidate markers for breast cancer.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (12), 3175-3180 (2017).</li><li>Li, P., Kaslan, M., Lee, S. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> Journal of Extracellular Vesicles. 4, 27031 (2015).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC+Transplantation+Improves+Osteopenia+via+Epigenetic+Regulation+of+Notch+Signaling+in+Lupus.">MSC Transplantation Improves Osteopenia via Epigenetic Regulation of Notch Signaling in Lupus.</a> Cell Metabolism. 22 (4), 606-618 (2015).</li><li>Deng, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+protection+by+mesenchymal+stem+cell+transplantation+identifies+exosomal+MiR-21+as+a+therapeutic+for+retinal+degeneration.">Photoreceptor protection by mesenchymal stem cell transplantation identifies exosomal MiR-21 as a therapeutic for retinal degeneration.</a> Cell Death and Differentiation. 28 (3), 1041-1061 (2021).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHED+aggregate+exosomes+shuttled+miR-26a+promote+angiogenesis+in+pulp+regeneration+via+TGF-beta/SMAD2/3+signalling.">SHED aggregate exosomes shuttled miR-26a promote angiogenesis in pulp regeneration via TGF-beta/SMAD2/3 signalling.</a> Cell Proliferation. 54 (7), 13074 (2021).</li><li>Qiu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+released+from+educated+mesenchymal+stem+cells+accelerate+cutaneous+wound+healing+via+promoting+angiogenesis.">Exosomes released from educated mesenchymal stem cells accelerate cutaneous wound healing via promoting angiogenesis.</a> Cell Proliferation. 53 (8), 12830 (2020).</li><li>He, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-derived+exosome+promotes+M2+polarization+and+enhances+cutaneous+wound+healing.">MSC-derived exosome promotes M2 polarization and enhances cutaneous wound healing.</a> Stem Cells International. 2019, 7132708 (2019).</li><li>Cheng, L., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutically+harnessing+extracellular+vesicles.">Therapeutically harnessing extracellular vesicles.</a> Nature Reviews Drug Discovery. 21 (5), 379-399 (2022).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Nielsen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle-associated+procoagulant+phospholipid+and+tissue+factor+activity+in+multiple+myeloma.">Extracellular vesicle-associated procoagulant phospholipid and tissue factor activity in multiple myeloma.</a> PLoS One. 14 (1), 0210835 (2019).</li><li>Zheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+vesicles+restore+liver+macrophage+homeostasis+to+counteract+type+2+diabetes.">Apoptotic vesicles restore liver macrophage homeostasis to counteract type 2 diabetes.</a> Journal of Extracellular Vesicles. 10 (7), 12109 (2021).</li><li>Gelibter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+storage+on+extracellular+vesicles:+A+systematic+study.">The impact of storage on extracellular vesicles: A systematic study.</a> Journal of Extracellular Vesicles. 11 (2), 12162 (2022).</li><li>Dehghani, M., Gulvin, S. 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EV'er dukker op til at spille en vigtig rolle i forskellige biologiske aktiviteter, herunder antigenpræsentation, transport af genetisk materiale, cellemikromiljømodifikation og andre. Desuden medfører deres brede anvendelse nye tilgange og muligheder for diagnosticering og behandling af sygdomme21. Implementering af terapeutiske anvendelser af EV'er er baseret på vellykket isolering og karakterisering. På grund af manglen på standardiserede isolerings- og rensningsmetoder og den lave ekstra...

Stamceller er udifferentierede pluripotente celler med evne til selvfornyelse og translationelt potentiale1. Mesenkymale stamceller (MSC'er) isoleres, dyrkes, udvides og renses let i laboratoriet, hvilket stadig er karakteristisk for stamceller efter flere passager. I de senere år har stigende dokumentation støttet det synspunkt, at MSC'er virker i en parakrin tilstand i terapeutisk brug 2,3. Især udskillelsen af ekstracellulære vesikler (EV'er) spiller en afgørende rolle i formidlingen af MSC'ernes biologiske funktioner. Som heterogene membranøse nanopartikler frigivet fra de fleste celletyper består EV'er af underkategorier kaldet exosomer (Exos), mikrovesikler (MV'er) og endnu større apoptotiske legemer 4,5. Blandt dem er Exos den mest undersøgte EV med en størrelse på 40-150 nm, som er af endosomal oprindelse og aktivt udskilles under fysiologiske forhold. MV'er dannes ved afgivelse direkte fra overfladen af celleplasmamembranen med en diameter på 100-1.000 nm, som er kendetegnet ved høj ekspression af phosphatidylserin og ekspression af overflademarkører af donorceller6. EV'er indeholder RNA, proteiner og andre bioaktive molekyler, som har lignende funktioner som modercellerne og spiller en væsentlig rolle i cellekommunikation, immunrespons og reparation af vævsskader7. MSC-EV'er er blevet bredt undersøgt som et kraftfuldt cellefrit terapeutisk værktøj i regenerativ medicin8.
Isolering og rensning af MSC-afledte elbiler er et almindeligt problem inden for forskning og anvendelse. På nuværende tidspunkt er differentiel og densitetsgradient ultracentrifugering9, ultrafiltreringsproces 10, immunomagnetisk separation11, molekylær eksklusionskromatograf 12 og mikrofluidisk chip13 almindeligt anvendte metoder til isolering og oprensning af elbiler. Med fordele og ulemper ved hver tilgang kan mængden, renheden og aktiviteten af indsamlede elbiler ikke opfyldes på samme tid14,15. I denne undersøgelse vises differentialcentrifugeringsprotokollen for isolering og karakterisering af EV'er fra dyrkede MSC'er i detaljer, hvilket har understøttet effektiv terapeutisk anvendelse 16,17,18,19,20. Tilpasningsevnen af denne metode til en række downstream-tilgange, såsom fluorescerende mærkning, lokal transplantation og systemisk injektion, er yderligere blevet eksemplet. Gennemførelsen af denne procedure vil imødekomme behovet for enkel og pålidelig indsamling og anvendelse af MSC-EV'er i translationel forskning.

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	<tbody>
		<tr>
			<td>10% povidone-iodine (Betadine)</td>
			<td>Weizhenyuan</td>
			<td>10053956954292</td>
			<td>Wound disinfection</td>
		</tr>
		<tr>
			<td>Calibration solution</td>
			<td>Particle Metrix</td>
			<td>110-0020</td>
			<td>Calibrate the NTA instrument</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Sigma</td>
			<td>53716-49-7</td>
			<td>Analgesic medicine</td>
		</tr>
		<tr>
			<td>Caudal vein imager&#160;</td>
			<td>KEW Life Science</td>
			<td>KW-XXY</td>
			<td>Caudal vein imager</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5418R</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Fatal bovine serum</td>
			<td>Corning</td>
			<td>35-081-CV</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Formvar/carbon-coated square mesh</td>
			<td>PBL Assay Science&#160;</td>
			<td>24916-25</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Zhongke Life Science</td>
			<td>Z8G5JBMz</td>
			<td>Post-treatment care of animals</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>StemCell</td>
			<td>7980</td>
			<td>Systemic injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science</td>
			<td>R510-22</td>
			<td>Animal anesthesia</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Alpha basic (1x)</td>
			<td>Gibco</td>
			<td>C12571500BT</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Nanoparticle tracking analyzer</td>
			<td>Particle Metrix</td>
			<td>ZetaView PMX120</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Meilunbio</td>
			<td>MA0015</td>
			<td>Resuspend EVs</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Procell Life Science</td>
			<td>PB180120</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Solarbio</td>
			<td>12501-23-4</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000224</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI26</td>
			<td>Labeling EVs</td>
		</tr>
		<tr>
			<td>Skin biopsy punch</td>
			<td>Acuderm</td>
			<td>69038-10-50</td>
			<td>Skin defects</td>
		</tr>
		<tr>
			<td>Software ZetaView</td>
			<td>Particle Metrix</td>
			<td>Version 8.05.14 SP7&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic equipment</td>
			<td>Grant</td>
			<td>v-0001-0005</td>
			<td>Water bath</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>HT7800</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>UCMSCs</td>
			<td>Bai&#39;ao&#160;</td>
			<td>UKK220201</td>
			<td>Commercially UCMSCs</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>XPN-100</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Ultrapure filtered water purification system</td>
			<td>Milli-Q</td>
			<td>IQ 7000</td>
			<td>Preparation of ultrapure water</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells

Ekstracellulære vesikler (EV'er) er heterogene membrannanopartikler frigivet af de fleste celletyper, og de anerkendes i stigende grad som fysiologiske regulatorer af organismehomeostase og vigtige indikatorer for patologier; I mellemtiden er deres enorme potentiale for at etablere tilgængelige og kontrollerbare sygdomsbehandlinger ved at dukke op. Mesenkymale stamceller (MSC'er) kan frigive store mængder EV'er i kultur, hvilket har vist løfte om at kickstarte effektiv vævsregenerering og lette omfattende terapeutiske anvendelser med god skalerbarhed og reproducerbarhed. Der er en stigende efterspørgsel efter enkle og effektive protokoller til indsamling og anvendelse af MSC-EV'er. Her leveres en detaljeret protokol baseret på differentiel centrifugering for at isolere og karakterisere repræsentative elbiler fra dyrkede humane MSC'er, exosomer og mikrovesikler til yderligere applikationer. Tilpasningsevnen af denne metode er vist for en række downstream-tilgange, såsom mærkning, lokal transplantation og systemisk injektion. Gennemførelsen af denne procedure vil imødekomme behovet for enkel og pålidelig indsamling og anvendelse af MSC-EV'er i translationel forskning.

MV'er og Exos fra dyrkede humane UCMSC'er isoleres efter den eksperimentelle arbejdsgang (figur 1). NTA-resultaterne viser, at størrelsen af Exos fra humane MSC'er varierer fra 40 nm til 335 nm med en topstørrelse på ca. 100 nm, og størrelsen af MV'er varierer fra 50 nm til 445 nm med en topstørrelse på 150 nm (figur 2). Morfologisk karakterisering af MSC-afledte Exos udviser en typisk kopform (figur 3). Elbiler mærkes effekt...

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Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Denne protokol beskriver differentiel centrifugering til isolering og karakterisering af repræsentative EV'er (exosomer og mikrovesikler) fra dyrkede humane MSC'er. Yderligere anvendelser af disse elbiler forklares også i denne artikel.

Isolering, karakterisering og terapeutisk anvendelse af ekstracellulære vesikler fra dyrkede humane mesenkymale stamceller

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Denne procedure adresserede behovet for enkel og pålidelig mesenkymal stamcelleafledt ekstracellulær vesikelopsamling og anvendelse i oversættelse af forskning. Indsamling af ekstracellulære vesikler ved differentiel centrifugering er enkel, let skalerbar og reproducerbar, hvilket vil være nyttigt for feltet. Ekstracellulære vesikler utydelige}fra utydelige}mesenkymale stamceller er en utydelig}anvendelse i moderne sygdomsbehandlinger.Jeg vil ikke være bange for fejl. Denne operation er enkel og nem at lære. Der er mange detaljer i dette enkle program, der bedre kan præsenteres via video.Til at begynde med skal du dyrke mesenkymale stamceller til mere end 90% sammenløb. Udskift dyrkningsmediet i hver skål med 8 ml suppleret alfa-MEM. Efter 48 timers dyrkning skal cellerne dyrkes jævnt for fuldt ud at samle dyrkningsmediet i 50 ml centrifugerør.Derefter centrifugeres kulturmediet ved 800 x g i 10 minutter ved fire grader Celsius. Supernatanten overføres til 1,5 ml koniske rør for at fjerne cellefragmenter og celleaffald. Supernatanten centrifugeres ved 16.000 x g i 30 minutter ved fire grader Celsius.Brug en pipette til at overføre suspensionen til ultracentrifugeglas. Derefter centrifugeres ved 150.000 x g i to timer ved fire grader Celsius. Mikrovesikelpelleten resuspenderes i 50 liter PBS pr. skål.Supernatanten kasseres, og resten, som er exosomer, opsamles. Resuspender exosomerne fra syv skåle af celler i 50 ml PBS. I et 15 ml rør fortyndes 1 uL ekstracellulære vesikler i 1.499 uL PBS og blandes.Start derefter den kompatible software til sporingsanalysatoren. Fyld prøvecellen med destilleret vand, og lad derefter instrumentet starte cellekontrollen. Kalibrer instrumentet med en forberedt standardopløsning.Sørg for, at partikelnummeret, der vises på softwaredetekteringsgrænsefladen, er mellem 50 og 400, helst omkring 200, og klik på okay. Skyl derefter prøvecellen med 5 ml destilleret vand. Før prøveanalyse skylles kanalen med 1 ml destilleret vand.Sørg for, at partikelnummeret, der vises på softwaredetekteringsgrænsefladen, er mindre end 10. I et 1,5 ml konisk rør resuspenderes de ekstracellulære vesikler med 250 ml PBS. I et andet 1,5 ml konisk rør fremstilles arbejdsløsningen af PKH26-farvestoffet.Bland de resuspenderede ekstracellulære vesikler med arbejdsløsningen. Lad det stå ved stuetemperatur i fem minutter, og tilsæt derefter 500 uL EV-udtømt FBS for at stoppe reaktionen. For mikrovesikler centrifugeres ved 16.000 x g i 30 minutter ved fire grader Celsius.Supernatanten kasseres. Tilsæt 1 ml PBS for at skylle resten. Der centrifugeres igen, og supernatanten kasseres for at fjerne det ubundne farvestof.For mikrovesikler centrifugeres ved 16.000 x g i 30 minutter ved fire grader Celsius. Rejsulfart resuspenderes med 200 liter PBS. Drop 20 uL suspension på diaset og observer det under et fluorescensmikroskop.Resuspender ekstracellulære vesikler i 200 uL PBS, og bland derefter med heparinopløsning i et volumenforhold på 10: 1. Placer musen i det kaudale venebilleddannelsessystem. Tryk på kontakten for at løfte håndtaget.Ved hjælp af en haleveneillustrator udføres en systematisk injektion af den ekstracellulære vesikelsuspension gennem halevenen. Analyse af partikelstørrelsesfordeling ved hjælp af NTA viste, at størrelsen af exosomer fra humane mesenkymale stamceller varierede fra 40 til 335 nm med en topstørrelse på ca. 100 nm. Størrelsen af mikrovesikler varierede også fra 50 til 445 nm med en topstørrelse på 150 nm.Morfologisk karakterisering af exosomer viste, at de udviste en typisk kopform. Ekstracellulære vesikler blev effektivt mærket af PKH26, som blev observeret af bruttovisningen som mærkede pellets såvel som ved fluorescerende mikroskopi. Operatøren bør være opmærksom under opsamlingen af supernatanter, da cellerne skal blæses jævnt for fuldt ud at opsamle de frigivne EV'er, der er tilbageholdt i mesenkymale stamcellers utydelige egenskaber.For at verificere injektionens succes kan der udføres in vivo-billeddannelse af biodistribution af fluorescensmærkede EV'er eller frosne sektioner af specifikke vævssteder for engraftment.

Research

JoVE Journal

Biology

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