Name: stem cell transplantation in an in vitro simulated ischemia/reperfusion model
Uploaded: 2011-11-05T14:00:00
Description: Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Questo lavoro è stato sostenuto da OTKA (Fondo di ricerca scientifica ungherese) D45933, T049621, TET (ungherese Fondazione Scienza e Tecnica) A4/04, Arg-17/2006 e SIN, Bolyai, borse di studio e Öveges TÁMOP 4.2.2-08 / 1 / KMR-2008-0004 e 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. Vorremmo ringraziare Gesztes William per la fornitura del voice-over.

1. Preparazione H9c2 cellule cardiomyoblast H9c2 cardiomyoblasts ratto sono stati ottenuti da ATCC (Wesel, Germania) e ampliato nel glucosio (4,5 g / L) DMEM contenente 10% di siero fetale bovino, 4 mM L-glutammina, 100 U / ml di penicillina e 100 mg / ml di streptomicina. La linea cellulare H9c2 mioblasti è derivato da cuore di ratto embrionale, è usato come un modello in vitro sia per muscolo scheletrico e cardiaco 8,9. Preparare 12 pozzetti di H9c2 ...

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Il protocollo è dimostrato un approccio in vitro alla questione molto più complessa di terapia con cellule staminali in infarto miocardico, con tutti i vantaggi e gli svantaggi di un tale modello. Ovviamente non può riflettere il complesso (ad esempio, immunologici) eventi che si svolgeranno durante e dopo l&#39;infarto del miocardio, ma può concentrarsi sugli effetti diretti delle cellule aggiunto sulle cellule postischemica. Gli effetti di ischemia simulata su H9c2 cardiomyoblasts molto dipende dal tempo di OGD, s...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> calceina-AM </td><td> Molecular Probes </td><td> L3224, C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> etidio omodimero-2 </td><td> Molecular Probes </td><td> L3224, E3599 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Vybrant DiD </td><td> Molecular Probes </td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> Tabella 1. Reagenti. <table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Commenti (opzionale) </td></tr><tr><td> Pecon sistema a celle di incubazione per microscopi Zeiss </td><td> Pecon GmbH </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> Tabella 2. Apparecchiature.

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

Protocolli di trapianto di cellule staminali stanno trovando la loro strada nella pratica clinica 1,2,3. Ottenere risultati migliori, rendendo i protocolli più robusto, e di trovare nuove fonti di cellule impiantabili sono al centro di recenti ricerche 4,5. Indagare l&#39;efficacia delle terapie cellulari non è un compito facile e nuovi strumenti sono necessari per indagare i meccanismi coinvolti nel processo di trattamento 6. Abbiamo progettato un protocollo sperimentale di ischemia / riperfusione in modo da consentire l&#39;osservazione di connessioni cellulari e dei meccanismi subcellulari anche durante l&#39;ischemia / riperfusione e dopo il trapianto di cellule staminali e per valutare l&#39;efficacia della terapia cellulare. H9c2 cellule cardiomyoblast sono stati collocati su piastre di coltura cellulare 7,8. Ischemia è stato simulato con 150 minuti in media glucosio libero con il livello di ossigeno inferiore allo 0,5%. Poi, i media normale e livelli di ossigeno sono state reintrodotte per simulare riperfusione. Dopo la deprivazione di ossigeno glucosio,le cellule danneggiate sono state trattate con il trapianto di midollo osseo umano etichettato cellule staminali mesenchimali aggiungendoli alla cultura. Cellule staminali mesenchimali sono preferiti negli studi clinici, perché sono facilmente accessibili con chirurgia mini-invasiva, facilmente espandibile e autologhe. Dopo 24 ore di co-coltura, le cellule sono state colorate con calceina e etidio-omodimero di distinguere tra cellule vive e morte. Questa configurazione ci ha permesso di indagare le connessioni intercellulari utilizzando la microscopia confocale a fluorescenza e di quantificare il tasso di sopravvivenza delle cellule postischemica mediante citometria di flusso. Microscopia confocale hanno mostrato le interazioni delle due popolazioni cellulari, come la fusione cellulare e la formazione di nanotubi intercellulari. Analisi di citometria a flusso ha rivelato 3 gruppi di cellule danneggiate, che può essere tracciata su un grafico e analizzati statisticamente. Queste popolazioni possono essere indagati separatamente e si possono trarre conclusioni su questi dati sull&#39;efficacia del simulatoapproccio terapeutico.

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Dimostriamo come impostare uno<em&gt; In vitro</em&gt; Ischemia / riperfusione modello e come valutare l&#39;effetto della terapia con cellule staminali su postischemica cellule cardiache.

Trapianto di cellule staminali in un In vitro Simulato ischemia / riperfusione Modello

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Research

JoVE Journal

Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

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The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

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