Name: 3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration
Uploaded: 2017-04-27T14:00:00
Description: Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

Forfatterne vil gerne anerkende QuinXell Technologies og CellD, især Lothar Grannemann og Dominique Ghozlan for deres hjælp med bioreaktoren. Vi takker Catherine Le Visage, der har givet os med pullulanet / dextran polysaccharid stilladser. Dette arbejde blev støttet af Den Europæiske Union (ERC-2014-cog projekt Matisse 648.779) og af AgenceNationalede Recherche (ANR), Frankrig (MagStem projekt ANR-11 SVSE5).

1. Konstruktion af de magnetiske enheder BEMÆRK: udstyr til cellepodning varierer afhængigt af anvendelsen (figur 1). Til dannelse af aggregater, er antallet af celler begrænset til 2,5 x 10 5 / aggregat, så de magnetiske tips skal være meget tynde (750 um i diameter). At pode 1,8 cm2 / 7 mm tykke stilladser, skal magneterne være større (3 mm i diameter) og vil sikre cellemigrering gennem porerne i stilladset. <ol><li> Konstruktion af en indret...

<ol>
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For det første fordi de teknikker, der præsenteres her stole på internalisering af magnetiske nanopartikler, en vigtigt spørgsmål er resultatet af nanopartiklerne, når de lokaliserer i cellerne. Det er korrekt, jern nanopartikler kan udløse potentiel toksicitet eller forringet differentieringskapacitet afhængig af deres størrelse, belægning, og eksponeringstiden 19, 22. Imidlertid har flere undersøgelser vist nogen indvirkning på cellulær fysiologi,...

Magnetiske nanopartikler anvendes allerede i klinikken som kontrastmidler til magnetisk resonans imaging (MRI), og deres terapeutiske anvendelser holde udvide. For eksempel er det for nylig blevet vist, at mærkede celler kan manipuleres in vivo under anvendelse af et eksternt magnetisk felt, og kan ledes og / eller opretholdes ved en defineret implantationsstedet 1, 2, 3. I regenerativ medicin, kan de anvendes til at konstruere organiserede væv in vitro 4, herunder karvæv 5, 6, 7, knogle 8, og brusk 9. Ledbrusk er nedsænket i en avaskulær miljø, hvilket gør reparationer af de ekstracellulære matrixkomponenter meget begrænsede, når skaden indtræffer. Af denne grund, researchfirmaerneh er i øjeblikket fokuseret på engineering af hyaline brusk udskiftninger, der kan implanteres i det defekte område. For at fremstille et autologt udskiftning, er nogle forskningsgrupper udforske anvendelsen af autologe chondrocytter som en cellekilde 10, 11, mens andre understreger kapaciteten af mesenkymale stamceller (MSC) til at differentiere til chondrocytter 12, 13. I tidligere undersøgelser er gengivet, valgte vi MSC, som deres knoglemarv prøveudtagning er forholdsvis enkel og kræver ikke det offer af sunde chondrocytter, som risikerer at miste deres fænotype 14. Et tidligt trin vigtigt at indlede den chondrogene differentiering af stamceller er deres kondens. Celleaggregater dannes almindeligvis ved anvendelse af enten centrifugering eller mikromassekultur 15; men disse kondensationsmetoder neither præsentere potentiale til at skabe celleklynger inden tykke stilladser eller potentialet til at kontrollere fusionen af ​​aggregater. I dette papir, beskriver vi en innovativ tilgang til kondenserende stamceller under anvendelse af MSC magnetisk mærkning og magnetiske tiltrækning. Denne teknik har vist sig at danne stillads-fri 3D konstruktioner via fusion af aggregater med hinanden for at opnå en millimeter-skala bruskvæv 9. Magnetisk podning af tykke og store skeletter har også givet mulighed for at øge størrelsen af ​​den konstruerede væv, designe en form lettere anvendelig til implantering, og diversificering af potentialet for kliniske anvendelser i brusk reparation. Her, vi detaljeret protokol for den magnetiske podning af MSC i porøse stilladser sammensat af naturlige polysaccharider, pullulan og dextran, stilladser tidligere blev anvendt til at begrænse stamceller 16, 17. Chondrogen differentiering var finally udført i en bioreaktor for at sikre løbende næringsstof og gas diffusion i matricen kerne af skeletter podet med en høj tæthed af celler. Ud over at give næringsstoffer, chondrogene vækstfaktorer og gas til cellerne, bioreaktoren tilbød mekanisk stimulation. Samlet set den magnetiske teknologi anvendes til at begrænse stamceller, kombineret med dynamisk modning i en bioreaktor, kan markant forbedre chondrogen differentiering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Iron&#160; oxide (maghemite) nanoparticules ( &#947;-Fe2O3)</td>
			<td>PHENIX - University Paris 6</td>
			<td>Made and given by C. M&#233;nager</td>
			<td>Mean diameter of 8.1 nm and negative surface charge</td>
		</tr>
		<tr>
			<td>Polysaccharide Pullulan/Dextran scaffolds</td>
			<td>LIOAD - University Nantes</td>
			<td>Made and given by C. Le Visage</td>
			<td>Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10M NaOH). Porosity: 185-205&#181;m; Thickness: 7mm; Surface area: 1.8cm2.</td>
		</tr>
		<tr>
			<td>TisXell Regeneration System</td>
			<td>QuinXell Technologies</td>
			<td>QX900-002</td>
			<td>Biaxial bioreactor with 500 mL culture chamber&#160;</td>
		</tr>
		<tr>
			<td>Cage for scaffolds: Histosette II M492&#160;</td>
			<td>VWR</td>
			<td>720-0909</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell (MSC)</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>Three independant batches have been used</td>
		</tr>
		<tr>
			<td>MSCGM BulletKit medium</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td>
		</tr>
		<tr>
			<td>DMEM with Glutamax I</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>RPMI medium 1640, no Glutamine</td>
			<td>Life Technologies</td>
			<td>31870-025</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>PBS w/o CaCl2 w/o MgCl2</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin (10.000U/mL)/Streptomicin (10.000&#181;g/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Premix Universal Culture Supplement (20x)</td>
			<td>Corning</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution 100mM</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphate</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td>Prepare the concentrated solution (25 mM) in distilled water extemporaneously</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma</td>
			<td>P5607</td>
			<td>Prepare the 175 mM stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>TGF-beta 3 protein 10&#181;g</td>
			<td>Interchim</td>
			<td>30R-AT028</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>VWR</td>
			<td>33615.268</td>
			<td>Prepare the 1M stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Pullulanase from Bacillus acidopullulyticus</td>
			<td>Sigma</td>
			<td>P2986</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextranase from Chaetomium erraticum</td>
			<td>Sigma</td>
			<td>D0443</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin RNA Extraction Kit</td>
			<td>Macherey-Nagel</td>
			<td>740955.5</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Life Technologies</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primer - Hexamer&#160;</td>
			<td>Promega</td>
			<td>C1181</td>
			<td>500 &#181;g/mL: Use diluted 1/2 and put 1 &#181;L per sample</td>
		</tr>
		<tr>
			<td>Recombinant RNAsin ribonuclease inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td>40 U/&#181;L: put 1 &#181;L per sample</td>
		</tr>
		<tr>
			<td>PCR nucleotide dNTP mix (10mM each)</td>
			<td>Roche</td>
			<td>10842321</td>
			<td></td>
		</tr>
		<tr>
			<td>SyBr Green PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4368708</td>
			<td></td>
		</tr>
		<tr>
			<td>Step One Plus Real-Time PCR System</td>
			<td>Life Technologies</td>
			<td>4381792</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution 10% neutral buffered</td>
			<td>Sigma</td>
			<td>HT5012</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT solution</td>
			<td>VWR</td>
			<td>361603E</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Sigma</td>
			<td>M32631</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue O</td>
			<td>VWR</td>
			<td>1.15930.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.310</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>VWR</td>
			<td>1.08323.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Pertex</td>
			<td>Histolab</td>
			<td>840</td>
			<td></td>
		</tr>
		<tr>
			<td>RPLP0 Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-TGCATCAGTAC 
			CCCATTCTATCAT-3&#39; ; 
			5&#39;-AAGGTGTAATC 
			CGTCTCCACAGA-3&#39;</td>
		</tr>
		<tr>
			<td>Aggrecan Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-TCTACCGCTGCGAGGTGAT-3&#39; ; 3&#39;-TGTAATGGAACACGATGCCTTT-5&#39;</td>
		</tr>
		<tr>
			<td>Collagen II Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-ACTGGATTGACCCCAACCAA-3&#39; ; 3&#39;-TCCATGTTGCAGAAAACCTTCA-5&#39;</td>
		</tr>
	</tbody>
</table>

3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration

Brusk engineering fortsat en udfordring på grund af vanskelighederne med at skabe en in vitro funktionel implantat ligner det native væv. En tilgang nylig undersøgt for udvikling af autologe udskiftninger involverer differentiering af stamceller til chondrocytter. For at påbegynde denne chondrogenese, en komprimeringsgrad af stamcellerne er påkrævet; derfor demonstrerede vi muligheden for magnetisk kondenserende celler, både inden tykke stilladser og stillads-fri, under anvendelse af miniaturiserede magnetiske kilder som celle attractors. Denne magnetiske fremgangsmåde blev også anvendt til at styre aggregat fusion og opbygge stillads-fri, organiseret, tredimensionale (3D) væv flere millimeter i størrelse. Ud over at have en forbedret størrelse, dannet af magnetisk drevne fusion væv præsenteret en signifikant stigning i ekspressionen af ​​kollagen II, og der blev observeret en lignende tendens til aggrecan-ekspression. Som det native brusk blev underkastet kræfter that påvirket sin 3D-struktur, var dynamisk modning også udført. En bioreaktor, der giver mekaniske stimuli blev anvendt til at dyrke de magnetisk podet stilladser over en periode på 21 dage. Bioreaktor modning i høj grad forbedret chondrogenese i cellularized stilladser; den ekstracellulære matrix opnået under disse betingelser var rig på kollagen II og aggrecan. Dette arbejde beskriver innovative potentiale magnetisk kondensering af mærkede stamceller og dynamisk modning i en bioreaktor til forbedret chondrogen differentiering, både scaffold-fri og inden polysaccharid stilladser.

For det første kan aggregater individuelt dannet ved anvendelse af mikro-magneter ved at afsætte 2,5 x 10 5 mærkede stamceller (figur 2A). Disse enkelt aggregater (~ 0,8 mm i størrelse) kan derefter fusioneres til større strukturer takket være sekventiel, magnetisk induceret fusion. For eksempel på dag 8 af chondrogen modning blev aggregater anbragt i kontakt i par til dannelse dubletter; firlinger blev samlet på dag 11 ved at sammenlægge 2 dubletter;...

Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Chondrogenese fra stamceller kræver finjustering dyrkningsbetingelserne. Her præsenterer vi en magnetisk tilgang til kondenserende celler, at et vigtigt skridt initiere chondrogenese. Derudover viser vi, at dynamisk modning i en bioreaktor gælder mekanisk stimulering til de cellulære konstruktioner og øger bruskagtig extracellulær matrixproduktion.

3D Magnetisk Stem Cell Aggregering og bioreaktor Modning til Brusk Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Research

JoVE Journal

Bioengineering

Use of Human Perivascular Stem Cells for Bone Regeneration

Brug af menneskelige perivaskulær stamceller til Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

Alginat mikrokapselsammensætning som en 3D-platform for Formering og differentiering af humane embryonale stamceller (embryonale) til forskellige linier

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Udarbejdelse af 3D Fibrin stilladser til Stem celledyrkningsapplikationer

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

Human primat Lung Decellularization og Recellularization bruge en specialiseret Large-orgel bioreaktor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Engineering 3D Cellularized kollagengeler for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D Hydrogel stillads til Artikulære chondrocyt Kultur og brusk Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Micropatterning and Assembly of 3D Microvessels

Micropatterning og Samling af 3D mikrokar

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting af brusk og huden væv analoger udnytter en roman passiv blanding enhed teknik for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Storstilet, automatiseret produktion af fedtafledte stamcellesfæroider til 3D-bioprint

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Billeddirigeret bioreaktor til generering af biomanirigeret luftvejsvæv

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...