Gli autori vorrebbero riconoscere QuinXell Technologies e CellD, in particolare Lothar Grannemann e Dominique Ghozlan per il loro aiuto con il bioreattore. Ringraziamo Catherine Le Visage, che ci ha fornito i pullulano / ponteggi polisaccaride destrano. Questo lavoro è stato sostenuto dall&#39;Unione Europea (progetto ERC-2014-Cog Matisse 648.779) e dalla AgenceNationalede la Recherche (ANR), la Francia (progetto MagStem ANR-11 SVSE5).

1. la costruzione dei dispositivi magnetici NOTA: I dispositivi utilizzati per semina delle cellule variano a seconda dell&#39;applicazione (Figura 1). A formare aggregati, il numero delle cellule è limitato a 2,5 × 10 5 / aggregato, così le punte magnetiche devono essere molto sottili (750 micron di diametro). Seminare i ponteggi 1,8 centimetri 2/7 mm di spessore, i magneti devono essere più grandi (3 mm di diametro) e garantiranno migrazione cellulare attraverso i pori del ponteggio. <ol><li> Costruzione di un dispositivo a micro-magneti per la formazione di aggregati (Figura 1A) <ol><li> Rendere microfori con un trapano 0,8 mm mediante piastre di alluminio (3 cm di diametro e spessore 6 mm). </li><li> Inserire una punta magnetica (750 micron di diametro) in ogni foro della piastra. </li><li> Posizionare questo disco sopra un magnete al neodimio permanente, che garantisce la magnetizzazione di saturazione. </li></ol></li><li> Costruzione di un dispositivo per ponteggio semina ( <strong&gt; Figura 1B) <ol><li> Tagliare polistirolo rigido in 2.4 cm 2 piazze. </li><li> Inserire 9 piccoli magneti (3 mm di diametro, 6 mm di lunghezza) equidistanti su una superficie di 1,6 cm 2. </li><li> Posizionare il dispositivo nel corso di un magnete al neodimio permanente. </li></ol></li></ol> 2. Stem Cell Labeling NOTA: Le cellule staminali sono state marcate con 0,1 mM nanoparticelle magnetiche per 30 min (2,6 ± 0,2 pg ferro / cella) a formare aggregati, mentre erano etichettati con 0,2 mM nanoparticelle magnetiche per 30 min (5 ± 0,4 pg ferro / cella) per seminare impalcature. Queste concentrazioni di nanoparticelle e tempi di incubazione sono stati utilizzati in precedenza e pubblicato per MSC e altre cellule 18, 19, ed è stato determinato che le nanoparticelle influenzato né vitalità cellulare né capacità di differenziazione MSC. La massa ferro incorporata dalle cellule staminali è stato misurato mediante single-cell magnetophoresis 19, 20. <ol><li> Cellule di coltura umane staminali mesenchimali (MSC) nella massima terreno di crescita delle cellule staminali mesenchimali (MSCGM) a 37 ° C e 5% di CO 2 fino vicino alla confluenza (~ 90%). </li><li> Preparare la soluzione di etichettatura magnetico miscelando 0,1 o 0,2 mM maghemite citrato rivestite con ossido di ferro (γFe 2 O 3; nucleo: 8 nm di diametro) in terreno 5A di McCoy privo di siero modificata a Roswell Park Memorial Institute (RMPI) senza glutammina e contenente 5 mM citrato di sodio. </li><li> Eliminare il mezzo, lavare le cellule con mezzo RPMI privo di siero senza glutammina, e aggiungere 10 ml di soluzione di ferro ossido di nanoparticelle a 150 cm 2 pallone di coltura, il volume minimo richiesto per coprire tutte le celle. </li><li> Incubare per 30 minuti a 37 ° C e 5% di CO 2 e quindi eliminare la soluzione di nanoparticelle. Sciacquare per 5 minuti con mezzo RPMI privo di siero senza glutammina per internalizzare il nanoparticles ancora attaccato alla membrana plasmatica. </li><li> Eliminare il medium RPMI e aggiungere 25 ml di completo media MSCGM per bombola. Incubare per una notte a 37 ° C e 5% CO 2. </li></ol> 3. semina delle cellule magnetiche <ol><li> Appena preparare il mezzo condrogenica usando Dulbecco Modified Eagle Medium (DMEM) high glucose con L-glutammina aggiungendo 50 mM di acido L-ascorbico 2-fosfato, 0,1 pM desametasone, 1 mM sodio piruvato, 0,35 mM L-prolina, 1% cultura universale supplemento contenente insulina, transferrina umana e acido selenioso (ITS-Premix), e 10 ng / mL di crescita trasformante fattore-beta 3 (TGF-β3). </li><li> Staccare le cellule magnetiche utilizzando 8 ml di 0,05% tripsina-EDTA a 150 cm-2 pallone di coltura e centrifugare le cellule dissociate a 260 xg per 5 min. Aspirare il terreno e contare le cellule risospese. </li><li> Posizionare una coltura cellulare con fondo piatto Petri (35 mm) al di sopra di entrambi i dispositivi magnetici. </li><li> per magnetically formare aggregati, aggiungere 3 ml di terreno condrogenica alla piastra di Petri e delicatamente depositare il minor volume possibile (non più di 8 mL) contenente 2,5 x 10 5 cellule marcate per aggregato (fino a 16 aggregati può essere depositato). Lasciare la piastra di Petri per 20-30 min senza spostarlo, permettendo di formare sferoidi, e quindi inserire il dispositivo completo, compresa la piastra di Petri contenente 16 aggregati, in incubatore a 37 ° C e 5% CO 2. </li><li> aggregati controllo forma seguendo lo stesso protocollo e sostituire il mezzo completo con il mezzo condrogenica senza TGF-β3. <ol><li> Per generare l&#39;aggregato costrutto 3D, mettere 2 aggregati in contatto il giorno 8 per formare 8 doppiette ed avviare la fusione. Il giorno 11, unire 2 doppiette per formare 4 quartine. Infine, fondere le 4 quadruplets il giorno 15 per ottenere la struttura finale. </li><li> Allo stesso tempo, formare aggregati centrifugando 2,5 × 10 5 cellule staminali etichettati a 26015; g per 5 min a 15 mL provette con 1,5 mL di terreno condrogenica con o senza TGF-β3 (per il campione e controllo, rispettivamente). </li></ol></li><li> Per magneticamente ponteggi seme, inserire ogni scaffold essiccato in una capsula di Petri. Utilizzare polisaccaridi scaffold porosi fatti di pullulano / destrano 21. Per ogni scaffold, diluire 2 x 10 6 cellule staminali marcate in 350 ml di mezzo condrogenica senza TGF-β3 e accuratamente pipetta le cellule sul ponteggio. <ol><li> Incubare per 5 min a 37 ° C per consentire la penetrazione cellulare completo entro l&#39;impalcatura e poi delicatamente aggiungere 3 mL di terreno condrogenica con o senza TGF-β3 (rispettivamente campione o controllo,) alla piastra di Petri. </li><li> Incubare il ponteggio cellularized sul suo dispositivo magnetico a 37 ° C e 5% di CO 2 per 4 giorni per consentire la migrazione cellulare attraverso i pori scaffold e confinamento. </li></ol></li><li> Allo stesso tempo, seminare ponteggi con 2 × 10 6 </sup&gt; cellule staminali etichettati seguendo lo stesso metodo e incubare senza il magnete per ottenere scaffold uniformemente seminato come controlli positivi. </li></ol> 4. differenziazione in condrociti NOTA: Dopo 4 giorni di incubazione, rimuovere i magneti e continuare la maturazione condrogenica sia in una capsula di Petri (condizioni statiche) o in un bioreattore (condizioni dinamiche). campioni di controllo negativi sono maturati in condizioni statiche di media condrogenica senza TGF-β3. <ol><li> In condizioni statiche, mantenere i ponteggi cellularized o aggregati della stessa piastra di Petri. Cambiare il medio condrogenica due volte a settimana per 21 giorni. </li><li> In condizioni dinamiche, preparare il bioreattore. <ol><li> Tagliare tubi in silicone alla lunghezza appropriata secondo il protocollo del produttore. </li><li> Autoclavare tutti materiali: 500 mL camera cultura, tubi, piastre rotanti 2 vie, e gabbie. </li><li> Posizionare le parti in un bioreattore microbio sterilistazione di sicurezza logico. Collegare il tubo per le 2 vie rotatori e alla camera di cultura seguendo le istruzioni del produttore. </li><li> Trasferire accuratamente i ponteggi cellularized in gabbie sterilizzati utilizzando una spatola sterile. Mettere 2 ponteggi per gabbia. Quando le gabbie sono pronte, inserirle nelle aghi del coperchio per impedire loro di muoversi durante un&#39;ulteriore rotazione. Riempire la camera di coltura con terreno condrogenica e chiuderlo con il coperchio contenente le gabbie. </li></ol></li><li> Accendere la pompa peristaltica per riempire il tubo con il mezzo condrogenica e per eliminare le bolle d&#39;aria. </li><li> Posizionare e fissare la camera riempita nel motore del bioreattore e accendere il computer, che controlla le rotazioni sia del braccio e della camera. </li><li> Applicare una velocità di rotazione di 5 rotazioni al minuto (rpm) sia sul braccio e la camera. Regolare la pompa peristaltica ad una portata di 10 rpm per l&#39;alimentazione continua dei ponteggi cellularized. </li></ol> NOTA: Prima di estrazione di RNA, digerire i ponteggi con una soluzione enzimatica. <ol><li> Preparare 1 mL di soluzione enzimatica aggiungendo 100 ml di pullulanasi (40 U / ml) e 50 ml di destranasi (60 mg / ml) a 850 ml di terreno DMEM privo di siero. </li><li> Lavare i ponteggi due volte con DMEM privo di siero, scartare il mezzo, e aggiungere 800 microlitri della soluzione enzimatica per ponteggio. Incubare per 15-30 minuti a 37 ° C sotto leggera agitazione. </li><li> Quando l&#39;impalcatura è completamente sciolto, trasferire la soluzione contenente le cellule ad una provetta da 1,5 mL, centrifugare a 300 xg per 10 min, aspirare accuratamente il mezzo, lavare due volte con sterile 1 × salina tamponata con fosfato (PBS), centrifugare a 300 g per 10 min, e risospendere le cellule in soluzione isolamento dell&#39;RNA. </li><li> Per estrarre l&#39;RNA dagli aggregati, posizionare i sferoidi nella soluzione di isolamento di RNA ecompletamente schiacciare usando un omogeneizzatore prima di eseguire l&#39;estrazione di RNA. </li><li> Isolare l&#39;RNA utilizzando un kit per l&#39;estrazione di RNA totale secondo le istruzioni del produttore. </li><li> Sintetizzare il DNA complementare da 400 ng di RNA totale utilizzando la trascrittasi inversa secondo le istruzioni del produttore, utilizzando 250 ng di primer casuali, 1 ml di miscela di dNTP (10 mM ciascuno), e 40 U / ml RNasi inibitore; il volume finale della reazione è di 20 microlitri. Al termine della reazione, aggiungere 80 ml di acqua distillata per ottenere un volume finale di 100 microlitri. </li><li> Per la reazione a catena della polimerasi (PCR) quantitativa, usare una miscela di PCR contenente un reagente fluorescente per quantificare l&#39;espressione relativa dei geni di interesse, come aggrecan (AGC) e collagene II (Col II), con 10 × diluito cDNA. Normalizzare i livelli di espressione genica con la Ribosomale proteina del gene di riferimento, di grandi dimensioni, P0 (RPLP0). Eseguire i calcoli con il 2 - formula ΔΔCT, Dove ΔΔCT = ΔCT condizione differenziata - significa ΔCT di condizione di controllo, e ogni ΔCT rappresenta la TC del gene di interesse - CT del gene di riferimento (RPLP0). </li><li> Determinare misurazioni statistiche come i valori medi ± l&#39;errore standard della media (SEM). Eseguire l&#39;analisi con n ≥ 2 esperimenti indipendenti. Utilizzare test t di analizzare le differenze statistiche tra pellets centrifugati e fusioni magnetici (* p &lt;0,05). Determinare la significatività con un test di Kruskal-Wallis (ANOVA non parametrico) per analizzare le differenze statistiche tra scaffold differenziati e con lo scaffold controllo (* p &lt;0,05). </li></ol> 6. analisi istologica <ol><li> Lavare i ponteggi cellularized o aggregati con sterile 1 × PBS, fissarli in formalina al 10% per 1 ora a temperatura ambiente, e risciacquare con 1 × PBS. </li><li> Rimuovere la PBS, incorporare i campioni in cuttin ottimalecomposto g temperatura (OCT), e congelarli in un bagno isopentano immersi in azoto liquido. Conservare il campione a -20 ° C. Tagliare i campioni con un criostato avere criosezioni 8 micron per gli aggregati o 12 micron per i ponteggi cellularized. </li><li> Macchiare le criosezioni con 0,5% toluidina soluzione blu per 2 min, risciacquo in acqua di rubinetto, disidratare con etanolo al 100%, chiarire con toluene, e montare i vetrini con un mezzo di montaggio per la microscopia ottica. </li></ol>

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	<li>Naumova, A. V., Modo, M., Moore, A., Murry, C. E., Frank, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+imaging+in+regenerative+medicine.">Clinical imaging in regenerative medicine.</a> Nat Biotechnol. 32 (8), 804-818 (2014).</li><li>Edmundson, M., Thanh, N. T., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+based+stem+cell+tracking+in+regenerative+medicine.">Nanoparticles based stem cell tracking in regenerative medicine.</a> Theranostics. 3 (8), 573-582 (2013).</li><li>Di Corato, R., 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=High-resolution+cellular+MRI:+gadolinium+and+iron+oxide+nanoparticles+for+in-depth+dual-cell+imaging+of+engineered+tissue+constructs.">High-resolution cellular MRI: gadolinium and iron oxide nanoparticles for in-depth dual-cell imaging of engineered tissue constructs.</a> ACS Nano. 7 (9), 7500-7512 (2013).</li><li>Xu, F., 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=Three-dimensional+magnetic+assembly+of+microscale+hydrogels.">Three-dimensional magnetic assembly of microscale hydrogels.</a> Adv Mater. 23 (37), 4254-4260 (2011).</li><li>Kito, 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=iPS+cell+sheets+created+by+a+novel+magnetite+tissue+engineering+method+for+reparative+angiogenesis.">iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis.</a> Sci Rep. 3, 1418 (2013).</li><li>Mironov, V., Kasyanov, V., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+in+vascular+tissue+engineering:+from+nanoscaffolding+towards+rapid+vessel+biofabrication.">Nanotechnology in vascular tissue engineering: from nanoscaffolding towards rapid vessel biofabrication.</a> Trends Biotechnol. 26 (6), 338-344 (2008).</li><li>Mattix, B. 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=Janus+magnetic+cellular+spheroids+for+vascular+tissue+engineering.">Janus magnetic cellular spheroids for vascular tissue engineering.</a> Biomaterials. 35 (3), 949-960 (2014).</li><li>Henstock, J., El Haj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+mechanotransduction+in+therapeutic+MSCs:+can+remotely+controlled+magnetic+nanoparticles+regenerate+bones?.">Controlled mechanotransduction in therapeutic MSCs: can remotely controlled magnetic nanoparticles regenerate bones?.</a> Regen Med. 10 (4), 377-380 (2015).</li><li>Fayol, D., 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=Use+of+magnetic+forces+to+promote+stem+cell+aggregation+during+differentiation,+and+cartilage+tissue+modeling.">Use of magnetic forces to promote stem cell aggregation during differentiation, and cartilage tissue modeling.</a> Adv Mater. 25 (18), 2611-2616 (2013).</li><li>Bartlett, W., 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=Autologous+chondrocyte+implantation+versus+matrix-induced+autologous+chondrocyte+implantation+for+osteochondral+defects+of+the+knee:+a+prospective,+randomised+study.">Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study.</a> J Bone Joint Surg Br. 87 (5), 640-645 (2005).</li><li>Batty, L., Dance, S., Bajaj, S., Cole, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation:+an+overview+of+technique+and+outcomes.">Autologous chondrocyte implantation: an overview of technique and outcomes.</a> ANZ J Surg. 81, 18-25 (2011).</li><li>Song, L., Baksh, D., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-based+cartilage+tissue+engineering:+cells,+scaffold+and+biology.">Mesenchymal stem cell-based cartilage tissue engineering: cells, scaffold and biology.</a> Cytotherapy. 6 (6), 596-601 (2004).</li><li>Boeuf, S., Richter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenesis+of+mesenchymal+stem+cells:+role+of+tissue+source+and+inducing+factors.">Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors.</a> Stem Cell Res Ther. 1 (4), 31 (2010).</li><li>Kock, L., van Donkelaar, C. C., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+functional+articular+cartilage:+the+current+status.">Tissue engineering of functional articular cartilage: the current status.</a> Cell Tissue Res. 347 (3), 613-627 (2012).</li><li>Schon, B. 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=Validation+of+a+high-throughput+microtissue+fabrication+process+for+3D+assembly+of+tissue+engineered+cartilage+constructs.">Validation of a high-throughput microtissue fabrication process for 3D assembly of tissue engineered cartilage constructs.</a> Cell Tissue Res. , (2012).</li><li>Robert, D., 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=Magnetic+micro-manipulations+to+probe+the+local+physical+properties+of+porous+scaffolds+and+to+confine+stem+cells.">Magnetic micro-manipulations to probe the local physical properties of porous scaffolds and to confine stem cells.</a> Biomaterials. 31 (7), 1586-1595 (2010).</li><li>Luciani, 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=Successful+chondrogenesis+within+scaffolds,+using+magnetic+stem+cell+confinement+and+bioreactor+maturation.">Successful chondrogenesis within scaffolds, using magnetic stem cell confinement and bioreactor maturation.</a> Acta Biomaterialia. 37, 101-110 (2016).</li><li>Wilhelm, C., Gazeau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+cell+labelling+with+anionic+magnetic+nanoparticles.">Universal cell labelling with anionic magnetic nanoparticles.</a> Biomaterials. 29 (22), 3161-3174 (2008).</li><li>Fayol, D., Luciani, N., Lartigue, L., Gazeau, F., Wilhelm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+magnetic+nanoparticle+aggregation+and+cellular+uptake:+a+precondition+for+efficient+stem-cell+differentiation+and+MRI+tracking.">Managing magnetic nanoparticle aggregation and cellular uptake: a precondition for efficient stem-cell differentiation and MRI tracking.</a> Adv Healthc Mater. 2 (2), 313-325 (2013).</li><li>Wilhelm, C., Gazeau, F., Bacri, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetophoresis+and+ferromagnetic+resonance+of+magnetically+labeled+cells.">Magnetophoresis and ferromagnetic resonance of magnetically labeled cells.</a> Eur Biophys J. 31 (2), 118-125 (2002).</li><li>Autissier, A., Le Visage, C., Pouzet, C., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+porous+polysaccharide-based+scaffolds+using+a+combined+freeze-drying/cross-linking+process.">Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process.</a> Acta Biomater. 6 (9), 3640-3648 (2010).</li><li>Singh, N., Jenkins, G. J. S., Asadi, R., Doak, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+toxicity+of+superparamagnetic+iron+oxide+nanoparticles+(SPION).">Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION).</a> Nano Reviews. 1, (2010).</li><li>Lee, J. H., Hur, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+formation+of+a+millimeter-scale+multicellular+spheroid+with+an+internal+cavity+from+magnetically+levitated+3T3+cells+that+ingested+iron+oxide-containing+microspheres.">Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.</a> Biotechnol Bioeng. 111 (5), 1038-1047 (2014).</li><li>Mattix, B., 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=Biological+magnetic+cellular+spheroids+as+building+blocks+for+tissue+engineering.">Biological magnetic cellular spheroids as building blocks for tissue engineering.</a> Acta Biomaterialia. 10 (2), 623-629 (2014).</li><li>Mazuel, F., 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=Massive+Intracellular+Biodegradation+of+Iron+Oxide+Nanoparticles+Evidenced+Magnetically+at+Single-Endosome+and+Tissue+Levels.">Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels.</a> ACS Nano. 10 (8), 7627-7638 (2016).</li><li>Kim, J. A., 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=High-throughput+generation+of+spheroids+using+magnetic+nanoparticles+for+three-dimensional+cell+culture.">High-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture.</a> Biomaterials. 34 (34), 8555-8563 (2013).</li><li>Shimizu, K., Ito, A., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell-seeding+into+3D+porous+scaffolds+by+use+of+magnetite+nanoparticles.">Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles.</a> J Biomed Mater Res B Appl Biomater. 77 (2), 265-272 (2006).</li><li>Sensenig, R., Sapir, Y., MacDonald, C., Cohen, S., Polyak, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle-based+approaches+to+locally+target+therapy+and+enhance+tissue+regeneration+in+vivo.">Magnetic nanoparticle-based approaches to locally target therapy and enhance tissue regeneration in vivo.</a> Nanomedicine (Lond). 7 (9), 1425-1442 (2012).</li><li>Waldman, S. D., Couto, D. C., Grynpas, M. D., Pilliar, R. M., Kandel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-axial+mechanical+stimulation+of+tissue+engineered+cartilage:+review.">Multi-axial mechanical stimulation of tissue engineered cartilage: review.</a> Eur Cell Mater. 13, 66-74 (2007).</li><li>Takahashi, I., 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=Compressive+force+promotes+sox9,+type+II+collagen+and+aggrecan+and+inhibits+IL-1beta+expression+resulting+in+chondrogenesis+in+mouse+embryonic+limb+bud+mesenchymal+cells.">Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.</a> J Cell Sci. 111 (14), 2067-2076 (1998).</li><li>Campbell, J. J., Lee, D. A., Bader, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compressive+strain+influences+chondrogenic+gene+expression+in+human+mesenchymal+stem+cells.">Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells.</a> Biorheology. 43, 455-470 (2006).</li><li>Vunjak-Novakovic, G., 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=Bioreactor+cultivation+conditions+modulate+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.</a> J Orthop Res. 17 (1), 130-138 (1999).</li></ol>

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In primo luogo, perché le tecniche qui presentate si basano su l&#39;internalizzazione delle nanoparticelle magnetiche, una questione importante è il risultato delle nanoparticelle, una volta che localizzano all&#39;interno delle cellule. È vero che le nanoparticelle di ferro possono scatenare tossicità potenziale o capacità di differenziazione ridotta seconda delle loro dimensioni, rivestimento, e il tempo di esposizione 19, 22. Tuttavia, numerosi studi ha...

nanoparticelle magnetiche sono già utilizzati in clinica, come agenti di contrasto per la risonanza magnetica (MRI), e le loro applicazioni terapeutiche tenere in espansione. Ad esempio, è stato recentemente dimostrato che le cellule marcate possono essere manipolati in vivo usando un campo magnetico esterno e possono essere orientati e / o mantenuti in un sito definito di impianto 1, 2, 3. In medicina rigenerativa, possono essere utilizzati per ingegnerizzare tessuti organizzati in vitro 4, tra tessuto vascolare 5, 6, 7, 8 osso, cartilagine e 9. La cartilagine articolare è immersa in un ambiente avascolare, le riparazioni dei componenti della matrice extracellulare molto limitati quando si verificano danni. Per questo motivo, research è attualmente focalizzata sulla progettazione di sostituzioni cartilagine ialina che possono essere impiantati nel sito del difetto. Per produrre una sostituzione autologo, alcuni gruppi di ricerca stanno studiando l&#39;utilizzo di condrociti autologhi come fonte cella 10, 11, mentre altri sottolineano la capacità delle cellule staminali mesenchimali (MSC) a differenziarsi in condrociti 12, 13. In studi precedenti ricapitolato qui, abbiamo scelto MSC, come il loro campionamento del midollo osseo è abbastanza semplice e non richiede il sacrificio di condrociti sani, che rischiano di perdere il loro fenotipo 14. Un primo passo essenziale per iniziare la differenziazione condrogenica delle cellule staminali è il loro condensazione. Aggregati cellulari sono comunemente formate utilizzando centrifugazione o cultura Micromass 15; Tuttavia, questi metodi di condensazione Neither presentare il potenziale per creare raggruppamenti cellulari all&#39;interno scaffold spessa né la possibilità di controllare la fusione degli aggregati. In questo documento, si descrive un approccio innovativo alla condensazione cellule staminali utilizzando marcatura magnetica MSC e l&#39;attrazione magnetica. Questa tecnica è stata dimostrata per formare costrutti 3D-free scaffold attraverso la fusione di aggregati tra loro per ottenere un millimetro scala tessuto cartilagineo 9. semina magnetico di scaffold spessi e grandi ha anche permesso la possibilità di aumentare la dimensione della ingegneria tessutale, realizzazione di una forma più facilmente utile per l&#39;impianto, e diversificare il potenziale per applicazioni cliniche di riparazione della cartilagine. Qui, si dettaglio il protocollo per la semina magnetico di MSC nella scaffold porosi costituiti da polisaccaridi naturali, pullulano, e destrano, scaffold precedentemente utilizzato per confinare cellule staminali 16, 17. differenziazione condrogenico era finally eseguita in un bioreattore per garantire continue dei nutrienti e diffusione del gas nel nucleo matrice degli scaffold seminati con un&#39;alta densità di cellule. Oltre a fornire le sostanze nutrienti, fattori di crescita condrogeniche e gas alle cellule, il bioreattore offerto stimolazione meccanica. Nel complesso, la tecnologia magnetica utilizzata per confinare le cellule staminali, in combinazione con la maturazione dinamica in un bioreattore, può notevolmente migliorare la differenziazione condrogenico.

<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

Ingegneria cartilagine rimane una sfida a causa delle difficoltà nel creare un impianto funzionale in vitro simile al tessuto nativo. Un approccio recentemente esplorato per lo sviluppo delle sostituzioni autologhe comporta la differenziazione delle cellule staminali in condrociti. Per avviare questo condrogenesi, un grado di compattazione delle cellule staminali è richiesta; quindi, abbiamo dimostrato la fattibilità di cellule magneticamente condensazione, sia all&#39;interno scaffold spessi e privo di scaffold, utilizzando miniaturizzati sorgenti di campo magnetico come attrattori cellulari. Questo approccio magnetico è stato utilizzato anche per guidare fusione aggregato e costruire privo di impalcatura, organizzato, tridimensionale (3D) i tessuti diversi millimetri di dimensione. Oltre ad avere una dimensione maggiore, il tessuto formato da fusione magnetica guidato presentato un significativo aumento dell&#39;espressione di collagene II, ed una tendenza simile è stato osservato per l&#39;espressione aggrecan. Come la cartilagine nativa è stato sottoposto a forze tcappello influenzato la sua struttura 3D, la maturazione dinamica è stata effettuata anche. Un bioreattore che fornisce stimoli meccanici stato usato per cultura scaffold magneticamente seminate su un periodo di 21 giorni. Bioreattore maturazione in gran parte migliorato chondrogenesis nelle impalcature cellularized; la matrice extracellulare ottenuto in queste condizioni era ricca di collagene II e aggrecano. Questo lavoro illustra il potenziale innovativo di condensazione magnetica delle cellule staminali etichettati e maturazione dinamica in un bioreattore per una migliore differenziazione condrogenica, sia-impalcatura libero e all&#39;interno polisaccaridi ponteggi.

In primo luogo, gli aggregati possono essere formati individualmente tramite microorganismi magneti depositando 2,5 x 10 5 cellule staminali marcate (Figura 2A). Questi singoli aggregati (~ 0,8 mm di dimensione) possono essere fusi in strutture più grandi grazie sequenziale, fusione indotta magneticamente. Per esempio, il giorno 8 di maturazione condrogenica, aggregati sono stati posti in contatto in coppia per formare doppietti; quartine sono state assemblat...

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

Condrogenesi da cellule staminali richiede la messa a punto delle condizioni di coltura. Qui vi presentiamo un approccio magnetico per condensare le cellule, un passo essenziale per iniziare condrogenesi. Inoltre, mostriamo che la maturazione dinamico in un bioreattore applica stimolazione meccanica ai costrutti cellulari e aumenta la produzione di matrice extracellulare cartilaginea.

3D Stem magnetica cellulare Aggregazione e bioreattore maturazione per la cartilagine Regeneration

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

3d-magnetic-stem-cell-aggregation-bioreactor-maturation-for-cartilage

Research

JoVE Journal

Bioengineering

9.7K Views.  UMR 7057 CNRS and University Paris Diderot.  Condrogenesi da cellule staminali richiede la messa a punto delle condizioni di coltura. Qui vi presentiamo un approccio magnetico per condensare le cellule, un passo essenziale per iniziare condrogenesi. Inoltre, mostriamo che la maturazione dinamico in un bioreattore applica stimolazione meccanica ai costrutti cellulari e aumenta la produzione di matrice extracellulare cartilaginea. 

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

Use of Human Perivascular Stem Cells for 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 a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

L&#39;uso di cellule staminali per la rigenerazione ossea perivascolari

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

Ricerca

Bioingegneria

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

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

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

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcapsule di alginato come piattaforma 3D per la propagazione e la differenziazione delle cellule staminali embrionali umane (hESC) per diverse linee

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

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 inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

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

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

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

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

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

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hydrogel Ponteggi per articolare condrociti, Cultura e Cartilagine Generation

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

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

Utilizzando adesivo Patterning per costruire dispositivi microfluidici di carta 3D

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e montaggio dei microvasi 3D

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 is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per Bioink Precellularization

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

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Modellazione della geometria delle colonie di cellule staminali embrionali umane su substrati conformi per controllare la meccanica a livello di tessuto

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...