Name: isolation and cultivation of mandibular bone marrow mesenchymal stem cells in rats
Uploaded: 2020-08-25T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats at JoVE.com

Vi takker for hjelpen fra Laboratoriet for digitalisert stomatologi og forskningssenter for kraniofaciale avvik ved Shanghai niende folkesykehus. Arbeidet med dette manuskriptet støttes av tilskudd fra National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit &Plateau Disciplines, SHIPM-mu-fondet fra Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], insentivprosjektet til innovasjonsteamet på høyt nivå for Shanghai Jiao Tong University School of Medicine. Og L.J. er forsker på Outstanding Youth Medical Talents, Shanghai "Rising Stars of Medical Talent" Youth Development Program og "Chen Xing" -prosjektet fra Shanghai Jiaotong University.

Alle eksperimentelle prosedyrer for dyr i denne artikkelen ble godkjent av Animal Care Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine.
1. Forberedelse
<ol><li>Bruk to 5 uker gamle mannlige Sprague Dawley rotter for eksperimentet.</li>
	<li>Steriliser alle instrumentene, inkludert nåleholdere, pinsett og saks ved høy temperatur eller nedsenket i 75% etanol i 10 minutter. MERK: Nedsenking av etanol bør ikke være for lang til å unngå cel...

<ol>
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L., McClung, G., Mammone, R. M., Ortved, K. 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Denne protokollen beskriver en metode for å isolere BMSCer fra rotte mandibles in vitro ved å kombinere hele benmargs overholdelse og fluorescerende celle sortering, som er en enkel og pålitelig måte å oppnå proliferative mBMSCs med sterk differensiering evne. Denne metoden kan foreløpig rense mBMSCer ved strømningscellesortering, men hvis det er høyere krav til cellehomogenitet, kan det være nødvendig med mer presise rensemetoder.
For tiden er det fire hovedteknikker som brukes til...

Benmarg mesenchymale stamceller (BMSCer) er ikke-hematopoietiske stamceller avledet fra benmarg som manifesterer sterk spredningsevne og multi-lineage differensieringspotensial1,2,3,4. Faktisk har BMSC-er blitt ansett som en ideell kandidat for beinvevsteknikk og regenerering helt siden de ble oppdaget. I årevis har iliac crest eller lange bein som tibia og lårben vært den vanligste kilden til BMSC for kraniofacial regenerering. Orofaciale BMSCer, for eksempel mandibulære BMSCer (mBMSCer), viser imidlertid noen forskjeller fra lange ben-BMSCer, for eksempel forskjellig embryonal opprinnelse og utviklingsmønster. Mandibler oppstår fra nevrale kamceller i nevroectoderm bakterielaget og gjennomgår intramembranøs ossifikasjon, mens aksiale og appendikulære skjeletter er fra mesodermet og gjennomgår endokondriell ossifikasjon. Videre har kliniske observasjoner og eksperimentelle dyrestudier konsekvent indikert at det er funksjonelle forskjeller mellom orofacial og iliac crest BMSCs5,6,7,8. Rapporter har vist at BMSC-er avledet fra kraniofacial bein som mandibel, maksillærben og alveolarben viste overlegen spredning, levetid og differensieringsevne enn de fra aksiale og appendikulære bein9. mBMSCs anses derfor å være de foretrukne ressursene for fremtidige terapeutiske anvendelser av kraniofaciale sykdommer som cherubisme, kjevetumor, osteoporose av kjeveben og periodontal vevsfeil10,11,12. For å forstå behandlingspotensialet i prekliniske eksperimenter, er det viktig å etablere en metode for raskt å isolere og dyrke mBMSCs in vitro.
I denne studien var målet å oppnå rensede mBMSCer ved hele benmargstilslutning og strømningscytometrisortering. Den anatomiske morfologien til rottemanidibel, tydelig observert gjennom mikroberegnet tomografi (Micro-CT) og histologiske seksjoner, viste at det trabekulære beinet i mandibelen var mellom snittet medullært rom og alveolarbenet. Benmargen fra trabekulære bein ble spylt for å oppnå mandibulære margceller, men cellene som ble dyrket på denne måten var ikke rene mBMSCer og var sannsynlig å bestå av flere typer celler med usikre potenser og forskjellige avstamninger som celler fra bein-, fett- og endotelceller13,14. Det neste trinnet i cellerensing var spesielt viktig. Flow cytometri filtrerer celler ved å gjenkjenne en kombinasjon av celleoverflateproteiner og har blitt mye vedtatt i berikelsen av mesenchymale stamceller. Cellehomogenitet er den største fordelen med strømningscytometri, men prosessen bestemmer ikke cellens levedyktighet og kan resultere i et begrenset celleutbytte. I denne studien ble P0 mBMSC oppnådd fra hele benmargstilslutning sortert etter strømningscytometri for å oppnå mBMSCer med høy renhet og sterk spredningskapasitet.
Denne studien introduserer en reproduserbar og pålitelig protokoll for isolasjon, kultur og differensiering av rotte mandibulære BMSCer ved hjelp av en kombinasjon av hele benmargs overholdelse og strømning cytometri sortering. Det er en pålitelig og praktisk metode for forskere i relaterte felt å bruke.

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA&#65288;1X&#65289;</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm culture dish</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>acutenaculum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adipogenic differentiation medium</td>
			<td>Cyagen biosciences inc.</td>
			<td>MUBMX-90031</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Beyotime Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Color Development Kit</td>
			<td>Beyotime Biotechnology</td>
			<td>C3206</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha-Minimum essential medium</td>
			<td>GE Healthcare HyClone Cell Culture</td>
			<td>SH30265.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti -CollagenII Rabbit pAb</td>
			<td>Abcam</td>
			<td>ab34712</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies against CD16/CD32</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antifade Mounting Medium with DAPI</td>
			<td>Beyotime Biotechnology</td>
			<td>P0131</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse/rat CD29 Antibody</td>
			<td>biolegend inc</td>
			<td>102215</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Esco</td>
			<td>AC2-4S8-CN</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (OX1), PE, eBioscience</td>
			<td>Invitrogen</td>
			<td>12-0461-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), FITC, eBioscience</td>
			<td>Invitrogen</td>
			<td>11-0900-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>cence</td>
			<td>L500</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondrogenesis differentiation medium</td>
			<td>cyagen biosciences inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM880</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet Staining Solution</td>
			<td>Beyotime Biotechnology</td>
			<td>C0121</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GE Healthcare HyClone Cell Culture</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Esco</td>
			<td>CCL-170B-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Magzol reagent(Trizol reagent)</td>
			<td>Magen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>micropipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osteogenic differentiation medium</td>
			<td>cyagen biosciences inc.</td>
			<td>MUBMX-90021</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline&#65288;1X&#65289;</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Kit</td>
			<td>TakaRa Bio Inc</td>
			<td>RR036A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P6556</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickBlock Blocking Buffer</td>
			<td>Beyotime Biotechnology</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR1 Premix</td>
			<td>TakaRa Bio Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Beyotime Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cultivation of mandibular bone marrow mesenchymal stem cells in rats

Her presenterer vi en effektiv metode for å isolere og dyrke mandibulær benmarg mesenchymale stamceller (mBMSCs) in vitro for raskt å oppnå mange celler av høy kvalitet for eksperimentelle krav. mBMSC kan være mye brukt i terapeutiske applikasjoner som vevsteknikkceller i tilfelle kraniofaciale sykdommer og kranio-maxillofacial regenerering i fremtiden på grunn av den utmerkede selvfornyelsesevnen og multi-lineage differensieringspotensialet. Derfor er det viktig å skaffe mBMSCer i stort antall.
I denne studien ble benmarg spylt fra mandible og primære mBMSCs ble isolert gjennom hele benmarg tilhenger dyrking. Videre ble CD29+CD90+CD45− mBMSC renset gjennom fluorescerende cellesortering. Den andre generasjonen rensede mBMSCer ble brukt til videre studier og viste potensial for å differensiere til osteoblaster, adipocytter og kondrocytter. Ved hjelp av denne in vitro-modellen kan man få et høyt antall proliferative mBMSCer, noe som kan lette studiet av de biologiske egenskapene, den påfølgende reaksjonen på mikromiljøet og andre anvendelser av mBMSCer.

Ved hjelp av denne protokollen holdt en stor andel celler seg til platen på den tredje dagen etter den første kulturen. Vanligvis, etter ytterligere 3-4 dager med kultur, nådde cellesamløpet til 70 til 80% (Figur 1B). Med fluorescerende cellesortering ble DAPI-CD29+CD90+CD45− mBMSC renset18,22, som utgjorde ca. 81,1 % i P0-cellene (figur 1C).
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Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Denne artikkelen presenterer en metode som kombinerer hele benmargstilslutning og strømningscytometrisortering for isolering, dyrking, sortering og identifisering av benmargmesenchymale stamceller fra rottemanidibler.

Isolasjon og dyrking av mandibulær benmarg Mesenchymale stamceller hos rotter

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

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