Questo lavoro è supportato da NHMRC Grant Program # 568969 (PSS) e la Facoltà di Medicina, Università di New South Wales, Stem Cell Initiative (KSS).

Tutte le procedure riportate di seguito vengono eseguite con tecniche asettiche all&#39;interno di una II classe biosicurezza Cabinet. Reagenti e attrezzature usati sono elencati nelle tabelle seguenti. 1. Preparazione del 1,1% alginato (w / v) <ol><li> Aggiungere 0,275 g di alginato di sodio purificato (elevato contenuto di acido glucuronico ≥ 60%, viscosità&gt; 200 mPa s, e endotossina ≤ 100 EU / g) in uno sterile tubo da 50 ml e aggiungere 25 ml di soluzione sterile 0,1% di gelatina preparata in precedenza (0,5 g gelatin/500 ml milli-Q H 2 O e sciolto in autoclave). </li><li> Vortex la provetta per circa 30 secondi per sciogliere parzialmente la polvere di alginato quindi posizionare il tubo in un mixer orbitale a 10 xg per il pernottamento (temperatura ambiente). </li><li> Aggiungere 2,778 ml del 9% sterile di NaCl (4,5 g di NaCl/50 ml Milli-Q H 2 O) a 25 ml di soluzione di alginato. Vortex la provetta per circa 30 secondi, seguito da centrifugazione a 95 xg per 5 min. </li><li> Conservare la soluzione di alginato a 4 °; C per la conservazione a breve termine (1-2 mesi) oppure a -20 ° C per la conservazione a lungo termine (circa un anno). </li></ol> 2. Preparazione di CaCl 2 Bath Precipitazioni <ol><li> Sciogliere 14.7 g di CaCl 2. 2H 2 O e 2,38 g di HEPES in 1 litro di Milli-Q H 2 O. </li><li> Regolare il livello di pH a 7,4. </li><li> Sterilizzare la soluzione con un filtro di 0,22 micron. </li><li> Bagno precipitazione può essere conservato a temperatura ambiente (6-12 mesi). </li></ol> 3. Preparazione della soluzione decapsulating <ol><li> In 500 ml di Phosphate Buffered Saline Dulbecco (D-PBS), aggiungere 50 ml di EDTA 0,5 M e 5 ml di HEPES 1M. </li><li> Sterilizzare usando un filtro di 0,22 micron o autoclave a 121 ° C per 20 min. </li><li> Conservare la soluzione decapsulating a temperatura ambiente (6-12 mesi). </li></ol> 4. Preparazione del siero di ricambio (SR) Medium <ol><li> Prima di incapsulamento, preparare media SR in anticipo, come dedescritto nella tabella di reagenti e attrezzature specifiche. </li></ol> 5. Preparazione di Inhibitor ROCK (Y-27632) <ol><li> Diluire Y-27.632 in polvere 0,1% albumina sierica umana (HSA) in D-PBS per ottenere una soluzione 5 mM. </li></ol> 6. Preparazione di hESC per l&#39;incapsulamento <ol><li> Pre-trattare le cellule con mezzi di coltura additivato con il 10 pM di inibitore ROCK (RI) per due ore a 37 ° C (riparo dalla luce). </li><li> Dopo il trattamento RI, lavare le cellule con D-PBS due volte e rimuovere le cellule da piastre di coltura enzimaticamente con accutase per 10 min a 37 ° C. </li><li> Raschiare delicatamente le cellule con il raschietto pipetta o cellulare e raccogliere in una provetta da 15 ml. Neutralizzare il accutase con il mezzo SR in rapporto 1:1. </li><li> Per preparare una sospensione di cellule singole, filtrare le cellule neutralizzati usando un filtro 40 um e raccogliere in un nuovo tubo da 50 ml centrifuga. </li><li> Calcolare il numero totale di cellule in soluzione con un emocitometro. </li&gt; <li> Centrifugare la sospensione cellulare 95 xg per 5 minuti e accuratamente scartare il surnatante dopo. Lavare le cellule con pre-riscaldata 0,9% NaCl. Centrifugare 95 xg per 5 minuti e scartare il surnatante. </li></ol> 7. Incapsulamento delle celle <ol><li> Preparare una siringa da 1 ml collegata a un tubo di plastica morbida 14G x 2 &quot;catetere IV. Questo viene utilizzato per aspirare la sospensione cellulare da caricare al pompa a siringa come mostrato nella Figura 1. </li><li> Risospendere le cellule con preriscaldata soluzione di alginato ad una densità di 1,25 milioni di cellule / ml alginato. Mescolare delicatamente con la siringa ed evitare di creare bolle. </li><li> Impostare il generatore tallone, pompa a siringa e il flussometro come mostrato nella Figura 1. Maggiori dettagli di questi dispositivi sono elencati nella Tabella di reagenti e attrezzature specifiche. </li><li> Con le cellule aspirato nella siringa, scartare il tubo di plastica del catetere IV e collegare la siringa alla macchina incapsulamento. Assicurarsi che vi sia spazio a 10 centimetri essere<html> tra la fine della macchina incapsulamento e il punto di raccolta, come mostrato nella Figura 1. </li><li> Incapsulare le cellule impostando la pompa a siringa a 20 ml / h, portata d&#39;aria a 8 L / min e una pressione di 100 kPa (la dimensione di capsule può essere modificata modificando la portata d&#39;aria). </li><li> Raccogliere le cellule incapsulate in una capsula di Petri (100 x 15 mm) riempita con 20 ml di pre-riscaldato bagno precipitazione per 7 minuti per stabilizzare le capsule. </li><li> Trasferire le cellule incapsulate aspirando piano in provette per centrifuga da 50 ml contenenti 20 ml di 0,9% NaCl. </li><li> Lasciare le capsule a depositarsi nella parte inferiore del tubo poi delicatamente scartare il surnatante. Ripetere il processo di lavaggio con NaCl 0,9%. </li><li> Risospendere le cellule incapsulate in preriscaldata terreno di coltura supplementato con RI (10 pM), il trasferimento in un pallone di coltura e incubate a 37 ° C / 5% CO 2. </li></ol> 8. Differenziazione delle hESC incapsulato in neuroni DA ve_content &quot;&gt; Il hESC incapsulato sono trattati con RI per 3 giorni prima della differenziazione. <ol><li> Seme della linea cellulare topo stromale, PA6 cellule ad una densità di 1,0 x 10 4 per cm 2 in 0,1% di gelatina rivestita T75 pallone e le condizioni PA6 cellule in mezzo differenziamento DA neurale (Tabella Materials) 24 ore prima della differenziazione. </li><li> Trasferire le capsule ad una provetta da centrifuga da 50 ml e consentire loro di depositarsi nella parte inferiore del tubo. </li><li> Eliminare il supernatante e risospendere le capsule in PA6 cella climatizzata di media differenziazione DA neurale. </li><li> Cultura del hESC incapsulato con il monostrato cellulare PA6 (9 x 10 6 hESC per 7,5 x 10 5 cellule PA6) per 28 giorni con un cambiamento dei media il giorno 4 e ogni altro giorno successivo (cambiare solo la metà del terreno di volta in volta). </li><li> Dopo 3 settimane in coltura con cellule PA6, integrare il mezzo DA differenziamento neurale con 100 ng / ml SHH e 100 ng / ml FGF8a per la settimana rimanente. </li&gt; </ol> 9. Decapsulation di hESC Encapsulated <ol><li> Aspirare le capsule in una provetta da centrifuga da 15 ml e consentire loro di depositano sul fondo della provetta. Poi scartare il surnatante con cura. </li><li> Lavare le capsule con D-PBS due volte. Lasciare le capsule si depositano sul fondo della provetta quindi rimuovere il supernatante. </li><li> Aggiungere 5 ml di soluzione decapsulating alle capsule. Mescolare la sospensione accuratamente tramite aspirazione prima di incubazione a temperatura ambiente per 4-5 min. </li><li> Centrifugare le cellule decapsulati a 95 xg per 3 min e scartare il surnatante. </li><li> Lavare il pellet di cellule con D-PBS seguita da centrifugazione a 95 xg per 3 min. Ripeti. </li><li> Le cellule decapsulate possono essere ulteriormente coltivate come monostrato o utilizzati per l&#39;analisi a valle. </li></ol> 10. Risultati rappresentativi Il diametro delle microcapsule è alginato di 400-500 micron. Il numero delle cellule all&#39;interno della capsula era estimated calcolando il numero totale di cellule diviso per il numero totale di capsule per corsa. Pertanto, a circa 5,0 x 10 4 cellule per capsula è stato stimato. Da questo, si presume che il numero massimo di cellule della capsula può contenere è di circa 1,0 x 10 5. La redditività di hESC incapsulato è&gt; 80% (Figura 2) come determinato utilizzando con diacetato carbossifluoresceina succinimidly estere (CFDA) / ioduro di propidio (PI) test. Abbiamo ottimizzato le condizioni di incapsulamento hESC diminuendo la concentrazione di alginato dal 2,2% al 1,1% e modificando il bagno precipitazione da cloruro di bario cloruro di calcio. Da queste condizioni, abbiamo dimostrato che solo le cellule che sono state incapsulate con alginato di calcio 1,1% potrebbe sopravvivere, proliferare e formare EBs in vitro 1. Per ottimizzare ulteriormente la condizione, gli effetti dei mezzi di coltura e RI, Y-27.632 sono stati esaminati. I dati presentati in Figura 2 e 3 dimostrano che prevente RId dissociazione apoptosi indotta e mantenuta la vitalità cellulare e la formazione di cluster promosso 1,6. Inoltre, la vitalità di hESC coltivate in incapsulato HFF-CM RI + era significativamente più alta rispetto agli altri gruppi senza supplementazione RI, ma questo non era significativamente diversa da incapsulato hESC coltivate in RI SR +. Analogamente, la proliferazione cellulare mediante saggio BrdU aumentata dal 25% al ​​75% come singole cellule sviluppate in cluster (Figura 3). Apoptosi da TUNEL rivelato che le singole cellule in microcapsule coltivate in mezzo SR erano apoptotico (dati non mostrati), mentre i cluster recuperati dalla HFF-CM erano negative per lo più TUNEL. In una certa misura, HFF-CM supplementate con bFGF anche promosso la sopravvivenza e la proliferazione delle hESC incapsulati in assenza di Y-27.632. Tuttavia, il trattamento con Y-27.632 prima (2 ore) e dopo l&#39;incapsulamento (per altri 4 giorni) marcatamente migliorata viabilità, la proliferazione e la formazione dei cluster hESC incapsulatoin microcapsule 1,1% di calcio alginato. In precedenza abbiamo dimostrato che hESC incapsulato può essere successo differenziate per endoderma definitivo 1. Qui, abbiamo esaminato l&#39;applicazione di incapsulamento delle cellule come una piattaforma 3D per differenziare hESC incapsulato in neuroni DA. hESC, che formavano corpi embrionali (EB) in capsule erano diretti differenziata e decapsulation alle condizioni descritte hanno mostrato una progressiva morfologia neuronale (Figura 4) dopo 2-3 giorni di coltura con oltre il 90% vitalità. Analisi di espressione genica ha mostrato una down-regulation dei marker pluripotenti, OCT4 mentre marker neuroprogenitor, PAX6 e marker DA neuronale, TH sono up-regolati dopo 7 giorni di differenziazione (Figura 5A). Colorazione immunofluorescenza ha rivelato che hESC differenziata erano PAX6-positivi (&gt; 80%), ma OCT4-negativi al giorno 7. Ulteriori hESC differenziato mostrato TH-positivi (&gt; 90%) neuroni dopo 21 giorni (Figura 5B). Western blot ha dimostratoun up-regolazione dell&#39;espressione dei TH dal giorno 14 (Figura 5C), mentre PAX6 espressione era down-regolato dopo giorno 21. In confronto, le cellule coltivate in ambiente bidimensionale (2D) in condizioni analoghe (RI esempio pretrattamento per 2 ore e RI post-trattamento per 3 giorni prima differenziazione) mantenuta un&#39;alta percentuale di cellule positive PAX6-(&gt; 80 %) per tutto il periodo di differenziazione e non erano così efficienti nella differenziazione di cellule TH-positive (&lt;60%) come in ambiente 3D fornito da incapsulamento (Figura 5A e C).

<ol>
	<li>Chayosumrit, M., Tuch, B., Sidhu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+microcapsule+for+propagation+and+directed+differentiation+of+hESCs+to+definitive+endoderm.">Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.</a> Biomaterials. 31, 505-514 (2010).</li><li>Dean, S. K., Yulyana, Y., Williams, G., Sidhu, K. S., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+encapsulated+embryonic+stem+cells+after+transplantation.">Differentiation of encapsulated embryonic stem cells after transplantation.</a> Transplantation. 82, 1175-1184 (2006).</li><li>Siti-Ismail, N., Bishop, A. E., Polak, J. M., Mantalaris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+benefit+of+human+embryonic+stem+cell+encapsulation+for+prolonged+feeder-free+maintenance.">The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance.</a> Biomaterials. 29, 3946-3952 (2008).</li><li>Dawson, E., Mapili, G., Erickson, K., Taqvi, S., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+stem+cell+differentiation.">Biomaterials for stem cell differentiation.</a> Adv. Drug Deliv. Rev. 60, 215-228 (2008).</li><li>Orive, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+encapsulation:+promise+and+progress.">Cell encapsulation: promise and progress.</a> Nat. Med. 9, 104-107 (2003).</li><li>Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Dang, S. M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled,+scalable+embryonic+stem+cell+differentiation+culture.">Controlled, scalable embryonic stem cell differentiation culture.</a> Stem Cells. 22, 275-282 (2004).</li><li>Drukker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+expression+of+MHC+proteins+in+human+embryonic+stem+cells.">Characterization of the expression of MHC proteins in human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U S A. 99, 9864-9869 (2002).</li><li>Calafiore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+Technical+Procedures+for+Microencapsulation+of+Human+Islets+for+Graft+into+Nonimmunosuppressed+Patients+With+Type+1+Diabetes+Mellitus.">Standard Technical Procedures for Microencapsulation of Human Islets for Graft into Nonimmunosuppressed Patients With Type 1 Diabetes Mellitus.</a> Transplantation Proceedings. 38, 1156-1157 (2006).</li><li>Cho, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3392-3397 (2008).</li><li>Vazin, T., Chen, J., Lee, C. T., Amable, R., Freed, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+stromal-derived+inducing+activity+in+the+generation+of+dopaminergic+neurons+from+human+embryonic+stem+cells.">Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells.</a> Stem Cells. 26, 1517-1525 (2008).</li></ol>

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Diversi studi che utilizzano cellule staminali embrionali di topo e hESC hanno dimostrato i benefici del sistema di coltura 3D in biomateriali e ingegneria dei tessuti 2,3. Abbiamo usato microcapsule di alginato di calcio come una piattaforma adatta 3D per studiare la propagazione hESC e la differenziazione rispetto a quanto hESC alginato di bario hanno mostrato significativa redditività superiore quando incapsulati in alginato di calcio di alginato di bario. Questo sistema consente inoltre una coltura ad al...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Note </td></tr><tr><td> Alginato (Pronova UP MVG) </td><td> NovoMatrix </td><td> 4200101 </td><td> elevato contenuto di acido glucuronico ≥ 60%, viscosità&gt; 200 mPa s, e endotossina &lt;100 EU / g </td></tr><tr><td> Gelatina </td><td> Sigma-Aldrich </td><td> G1890-100G </td><td></td></tr><tr><td> 0,9% NaCl </td><td> Baxter Healthcare </td><td> AHF7123 </td><td></td></tr><tr><td> Tipo J1 tallone generatore </td><td> Nisco engineering Inc </td><td> SPA-0447 </td><td></td></tr><tr><td> Multi-Phaser pompa siringa </td><td> New Era Pump Systems Inc </td><td> Modello NE-1000 </td><td></td></tr><tr><td> Ezi-Flow Flussimetro Medical </td><td> Gascon Sistemi</td><td> G0149 </td><td></td></tr><tr><td> Y-27.632 </td><td> Merck </td><td> 688000 </td><td></td></tr><tr><td> Albumina sierica umana </td><td> Sigma-Aldrich </td><td> A4327-1G </td><td></td></tr><tr><td> Accutase </td><td> Millipore </td><td> SCR005 </td><td></td></tr><tr><td> 14G x 2 &quot;catetere IV </td><td> Terumo </td><td> SR-OX1451C </td><td></td></tr><tr><td> Knockout-DMEM </td><td> Invitrogen </td><td> 10829-018 </td><td> Per SR medio (basale) </td></tr><tr><td> -I Glutamax </td><td> Invitrogen </td><td> 35050-061 </td><td> Per mezzo SR (2 mM) </td></tr><tr><td> Knockout sostituzione Siero </td><td> Invitrogen </td><td> 10828-028 </td><td> Per SR mezzo (20%) </td></tr><tr><td> Penicillina-Streptomicina </td><td> Invitrogen </td><td> 15070063 </td><td> Per mezzo SR (2,5 U / ml) </td&gt; </tr><tr><td> Insulin-transferrina-Selenio (ITS) </td><td> Invitrogen </td><td> 41400045 </td><td> Per SR media (1x) </td></tr><tr><td> β-mercaptoetanolo </td><td> Invitrogen </td><td> 21985-023 </td><td> Per mezzo di SR (0,1 mm) </td></tr><tr><td> MEM NEAA Solution </td><td> Invitrogen </td><td> 11140-050 </td><td> Per mezzo SR (5 mM) </td></tr><tr><td> Glasgow Media minimi essenziali </td><td> Invitrogen </td><td> 11710035 </td><td> Per mezzo di differenziazione DA neurale (basale) </td></tr><tr><td> Knockout sostituzione Siero </td><td> Invitrogen </td><td> 10828-028 </td><td> Per mezzo di differenziazione DA neurale (10%) </td></tr><tr><td> Sodio piruvato </td><td> Invitrogen </td><td> 11360070 </td><td> Per mezzo di differenziazione DA neurale (1 mM) </td></tr><tr><td> MEM NEAA Solution </td><td> Invitrogen </td><td> 11140-050 </td><td> Per DA ndifferenziazione media EURAL (0,1 mm) </td></tr><tr><td> β-mercaptoetanolo </td><td> Invitrogen </td><td> 21985-023 </td><td> Per mezzo di differenziazione DA neurale (0,1 mm) </td></tr><tr><td> Sonic hedgehog (SHH) </td><td> R &amp; D Systems </td><td> 1314-SH-025/CF </td><td> Per differenziazione DA neurale (100 ng / ml) </td></tr><tr><td> Fattore di crescita dei fibroblasti 8a (FGF8a) </td><td> R &amp; D Systems </td><td> 4745-F8-050 </td><td> Per differenziazione DA neurale (100 ng / ml) </td></tr></tbody></table>

alginate microcapsule as a 3d platform for propagation and differentiation of human embryonic stem cells (hesc) to different lineages

Cellule staminali embrionali umane (hESC) stanno emergendo come una fonte alternativa per la terapia di sostituzione cellulare dal momento che possono essere espanse in coltura indefinitamente e differenziati a tutti i tipi di cellule nel corpo. Vari tipi di biomateriali sono stati utilizzati anche in colture di cellule staminali per fornire un microambiente che imita la nicchia delle cellule staminali 1-3. Quest&#39;ultimo è importante per promuovere cellula-cellula interazione, proliferazione cellulare, differenziazione e in lignaggi specifici nonché l&#39;organizzazione fornendo un tessuto tridimensionale (3D) ambiente 4 come l&#39;incapsulamento. Il principio della cella incapsulamento implica l&#39;intrappolamento delle cellule viventi all&#39;interno dei confini di membrane semipermeabili in colture 3D 2. Queste membrane per lo scambio di nutrienti, ossigeno e stimoli attraverso le membrane, mentre gli anticorpi e le cellule immunitarie dall&#39;host che sono maggiori delle dimensioni dei pori sono esclusi capsula 5. Qui, si preha inviato un approccio alla cultura e differenziare hESC neuroni DA in un microambiente 3D utilizzando microcapsule di alginato. Abbiamo modificato le condizioni di coltura da 2 a migliorare la redditività di hESC incapsulato. Abbiamo precedentemente dimostrato che l&#39;aggiunta di gp160-Rho-associata coiled-coil chinasi (ROCK) inibitore, Y-27.632 e fetale umano fibroblasti terreno condizionato sostituzione siero (HFF-CM) alla piattaforma 3D significativamente migliorato la redditività di hESC incapsulato in cui le cellule espresso definitive geni marcatori endoderma 1. Abbiamo utilizzato questa piattaforma 3D per la propagazione della differenziazione hESC ed efficiente ai neuroni DA. Analisi di espressione proteica e genica dopo la fase finale di differenziazione neuronale DA mostrato un aumento dell&#39;espressione della tirosina idrossilasi (TH), un marcatore per DA neuroni,&gt; 100 pieghe dopo 2 settimane. Abbiamo ipotizzato che la nostra piattaforma 3D con microcapsule di alginato può essere utile per studiare la proliferazione e la differenziazione direttadi hESC a vari lignaggi. Questo sistema 3D permette anche la separazione di cellule feeder dal hESC durante il processo di differenziazione e ha anche un potenziale di immuno-isolamento durante il trapianto in futuro.

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

Abbiamo ottimizzato una tecnica di microincapsulazione come una piattaforma efficace 3D per la propagazione e la differenziazione delle cellule staminali embrionali per endoderma e dopaminergici (DA) neuroni. Esso fornisce inoltre l&#39;opportunità di immuno-isolamento di cellule dall&#39;host durante il trapianto. Questa piattaforma può essere adattato per altri tipi di cellule.

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 ...

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

16.2K Views.  The University of New South Wales. Abbiamo ottimizzato una tecnica di microincapsulazione come una piattaforma efficace 3D per la propagazione e la differenziazione delle cellule staminali embrionali per endoderma e dopaminergici (DA) neuroni. Esso fornisce inoltre l'opportunità di immuno-isolamento di cellule dall'host durante il trapianto. Questa piattaforma può essere adattato per altri tipi di cellule.

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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotazione Cultura sistemi cellulari per colture cellulari umane: cellule del trofoblasto umano come modello

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Sviluppo, di espansione, e<em&gt; In vivo</em&gt; Monitoraggio delle cellule NK umane da cellule staminali embrionali umane (hESC) ed e cellule staminali pluripotenti indotte (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Ordinazione di singole cellule e singola embrioni in 3D confinamento: Un nuovo dispositivo per l&#39;alta Screening contenuti

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered alginato costrutti: Una piattaforma per la cooperazione culturale delle popolazioni cellulari eterogenee

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Un metodo di coltura per mantenere le cellule staminali ematopoietiche umane quiescenti

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

Microscopia multifotonica intravitale in tempo reale per visualizzare trattamenti a ultrasuoni e microbolle focalizzati per aumentare la permeabilità della barriera emato-encefalica

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

In vitro Induzione di cellule staminali della polpa dentale umana verso lignaggi pancreatici

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...