Questo lavoro è stato sostenuto da un finanziamento della National Institute of Arthritis e muscolo-scheletrico e malattie della pelle: 3R01AR054458-02S2.

1. Giorno 1 1) Piastra cellule <ol><li> HMSC piastra in un pallone T75 ad una confluenza del 80% almeno 18-24 ore prima dell&#39;etichettatura. Fare riferimento alla Tabella 1 per le istruzioni per le navi alternativo. </li></ol> 2. Giorno 2 2) Preparare la soluzione di etichettatura. Questa preparazione si etichetta un (1) T75 fiasco al 80% confluenza con una concentrazione di 400 mg Fe / ml. Fare riferimento alla Tabella 1 per le istruzioni per le navi alternativo. <ol><li> Creare una soluzione (soluzione 1) mescolando 1 ml di siero-free media e 93,1 l di ferumoxytol (magazzino: 30 mg / ml) in un tubo da 15 ml. </li><li> Creare una seconda soluzione (soluzione 2) mescolando 1 ml di siero privo di mezzi di comunicazione e 7 ml di solfato di protamina (magazzino: 10 mg / ml) in un secondo tubo di 15 ml. </li><li> Consentire ad ogni soluzione a riposare per 5 minuti. </li><li> Aggiungere le soluzioni 1 e 2 insieme. Mescolare delicatamente la nuova soluzione di etichettatura. Lasciare che la soluzione a riposare per 5 minuti per consentire USPIO-procomplessi solfato di istamina a formare. </li><li> Aggiungere 5 ml di siero-multimediale gratuito al misto 2 ml di SPIO / soluzione di solfato di protamina per un volume finale di 7 ml. </li></ol> 3) Preparare le cellule per l&#39;etichettatura <ol><li> Aspirare i media dalle cellule. </li><li> Lavare delicatamente le cellule una volta con 2-3 ml di pre-riscaldato Mg / Ca-free D-PBS o senza siero media per lavare via residuale le proteine ​​del siero e dei media o altri componenti che potrebbero mettere in pericolo agente di assorbimento di contrasto e di efficienza etichettatura. Aspirare il liquido di risciacquo. </li></ol> 4) Etichetta cellule <ol><li> Aggiungere il completo 7 ml di soluzione di etichettatura per le cellule. </li><li> Cellule posto in un incubatore (37 ° C / 5% di CO 2) e permettono alle cellule di incubare nella soluzione di etichettatura per 4 ore. </li><li> Aggiungere 700 ml di FCS alla soluzione di etichettatura delle cellule per ottenere una concentrazione finale del siero del 10%. Siero viene aggiunto a ristabilire l&#39;ambiente arricchito di cui sono abituati le cellule, e per assistere in minimizing morte delle cellule che potrebbe derivare da un brusco passaggio a 24 ore di esposizione ad un ambiente privo di siero. </li><li> Permettono alle cellule di incubare con la soluzione di etichettatura per altre 20 ore. </li></ol> 3. 3 ° giorno 5) Preparare le cellule etichettate <ol><li> Aspirare la soluzione di etichettatura da parte delle cellule. </li><li> Lavare delicatamente le cellule con 2-3 ml di pre-riscaldato Mg / Ca-free D-PBS. Aspirare il PBS. E &#39;importante utilizzare Mg / Ca-free PBS come Mg e Ca potrebbe compromettere l&#39;efficienza della tripsina nella fase successiva. </li><li> Aggiungere 2 ml di pre-riscaldato tripsina 0,05% per le cellule e inclinare il pallone avanti e indietro per garantire l&#39;intera superficie del pallone è ricoperta da un sottile strato di tripsina. Mettere le cellule in un incubatore (37 ° C / 5% di CO 2) e permettono alle cellule di riposare per 5-7 minuti o fino a quando le cellule iniziano a staccarsi dal piatto. Potrebbe essere necessario per confermare il distacco al microscopio. Battere delicatamente i lati del pallone, se necessario, per facilitate superficie cellulare distacco. </li><li> Aggiungere 4 ml di supporti completi pre-riscaldato al pallone per neutralizzare la tripsina. Lavare delicatamente il pallone pipettando la tripsina / media / soluzione cellulari su e giù diverse volte. Raccogliere l&#39;intera soluzione in un tubo da 15 ml. Centrifugare la soluzione delle cellule a 400 RCF per 5 minuti. </li><li> Attentamente aspirare il surnatante senza disturbare il pellet. Risospendere le cellule in 5 ml di mezzi di comunicazione (contenente siero o siero-free). Centrifugare la soluzione delle cellule a 400 RCF per 5 minuti. </li><li> Ripetere il lavaggio delle cellule descritto al punto 5.5. In totale, le cellule saranno lavata tre volte per rimuovere residui, free agent di contrasto nei media e sulla superficie delle cellule. </li><li> Contare le celle a questo punto per raggiungere la conta delle cellule più accurate, come le cellule verranno persi durante il lavaggio precedente. Eseguire un test di vitalità delle cellule. Le cellule sono ora pronte per la successiva analisi e di sperimentazione o applicazione. RM può essere eseguita con T1-w e<html> T2-w parametri, perché anche se ferumoxytol è un T2-w mezzo di contrasto, ferumoxytol espone anche effetti T1. Rappresentante T2-w parametri RMN che possono essere utilizzati per l&#39;immagine cellule ferumoxytol etichettati comprendono: le sequenze FSE o SE_Multislice con un TR di 2000-2500 ms e un TE di 60-80 ms. Per acquisire un T1-w immagine, diminuire il valore TE su un FSE o sequenza SE_Multislice o utilizzare una sequenza GRE con un alto e basso TR TE. Figura 4 mostra un potenziale di applicazioni per l&#39;imaging in vivo di cellule staminali etichettati. </li></ol> 4. Rappresentante dei risultati: Cellule marcate dimostrare un effetto di contrasto significativo oscuramento o negativo su immagini T2-pesate MR e un effetto di luminosità o contrasto positivo sul T1 pesate MR (Figura 2). Longitudinale RM e test di vitalità eseguite 5 giorni dopo l&#39;etichettatura dimostrato alcun segnale significativo MR e nessun impatto significativo sulla viabilità rispetto ai controlli senza etichetta (dati non riportati). jove_content &quot;&gt; cellule staminali con etichetta possono essere successivamente differenziate in vari tipi di cellule o iniettata per via endovenosa per un esame più vivo. <img alt="Figura 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> Figura 1. Schematica linea del tempo per l&#39;etichettatura di cellule staminali con nanoparticelle di ossido di ferro, ferumoxytol. <img alt="Figura 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> Figura 2. Immagini RM di sezioni sagittali thrrough tubi eppendorf con le cellule in una pallina di cellule. A) senza etichetta controlli. B) le cellule etichetta dimostrando un significativo T2 o negativo effetto di contrasto pesata in T2 FSE o SE_Multislice imaging e un T1 o positivo effetto di contrasto sulla sequenza T1 GRE. <img alt="Figura 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> Figura 3. Sagittale sezioni dei tubi eppendorf con le cellule in cellule PELlascia. Un minimo di 10.000 cellule ferumoxytol etichettati possono essere rilevati tramite T2-w RM. <img alt="Figura 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> Figura 4. Visualizzazione MR di ferumoxytol marcato le cellule staminali iniettate nei ventricoli cerebrali murine. A) assiale, l&#39;immagine di MR barin murini senza iniezione di USPIO marcato le cellule staminali. B) assiale, C) coronale, e D) sagittale immagini RM raffigurante il T2, effetto negativo di contrasto di USPIO marcata con cellule staminali (frecce bianche) iniettato in un cervello murino. <img alt="Tabella 1" src="/files/ftp_upload/3482/3482table1.jpg" /> Tabella 1. Adattamenti Quantità per l&#39;etichettatura delle cellule nei vasi alternativo.

<ol>
	<li>Narsinh, K. H., Plews, J., Wu, 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=Comparison+of+human+induced+pluripotent+and+embryonic+stem+cells:+fraternal+or+identical+twins?.">Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical twins?.</a> Mol Ther. 19, 635-638 (2011).</li><li>Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+cell+tracking:+clinical+studies.">In vivo MRI cell tracking: clinical studies.</a> AJR. Am. J. Roentgenol. 193, 314-325 (2009).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+Iron+Oxide+Nanoparticles+for+Non-Invasive+in+vivo+Tracking+with+MR+Imaging.">Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging.</a> J. Vis. Exp. (13), e685-e685 (2008).</li><li>Tallheden, T., Nannmark, U., Lorentzon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+imaging+of+magnetically+labeled+human+embryonic+stem+cells.">In vivo MR imaging of magnetically labeled human embryonic stem cells.</a> Life. Sci. 79, 999-1006 (2006).</li><li>Jung, C. W., Jacobs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+properties+of+superparamagnetic+iron+oxide+MR+contrast+agents:+ferumoxides,+ferumoxtran,+ferumoxsil.">Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.</a> Magn. Reson. Imaging. 13, 661-674 (1995).</li><li>Coyne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferumoxytol+for+treatment+of+iron+deficiency+anemia+in+patients+with+chronic+kidney+disease.">Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease.</a> Expert. Opin. Pharmacother. 10, 2563-2568 (2009).</li><li>Li, Z., Suzuki, Y., Huang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reporter+gene+and+iron+particle+labeling+for+tracking+fate+of+human+embryonic+stem+cells+and+differentiated+endothelial+cells+in+living+subjects.">Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects.</a> Stem Cells. 26, 864-873 (2008).</li><li>Metz, S., Bonaterra, G., Rudelius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+human+monocytes+to+phagocytose+approved+iron+oxide+MR+contrast+agents+in+vitro.">Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro.</a> Eur. Radiol. 14, 1851-1858 (2004).</li><li>Nedopil, A., Klenk, C., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+signal+characteristics+of+viable+and+apoptotic+human+mesenchymal+stem+cells+in+matrix-associated+stem+cell+implants+for+treatment+of+osteoarthritis.">MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.</a> Invest. Radiol. 45, 634-640 (2010).</li><li>Kraitchman, D. L., Heldman, A. W., Atalar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+magnetic+resonance+imaging+of+mesenchymal+stem+cells+in+myocardial+infarction.">In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction.</a> Circulation. 107, 2290-2293 (2003).</li><li>Stuckey, D. J., Carr, C. A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+particles+for+noninvasive+monitoring+of+bone+marrow+stromal+cell+engraftment+into,+and+isolation+of+viable+engrafted+donor+cells+from,+the+heart.">Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.</a> Stem Cells. 24, 1968-1975 (2006).</li><li>Henning, T. D., Sutton, E. J., Kim, 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+influence+of+ferucarbotran+on+the+chondrogenesis+of+human+mesenchymal+stem+cells.">The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells.</a> Contrast. Media. Mol. Imaging. 4, 165-173 (2009).</li><li>Arbab, A. S., Yocum, G. T., Kalish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+Ferumoxides+Labeled+Human+Mesenchymal+Stem+Cells+in+Cartilage+Defects.">Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects.</a> J. Vis. Exp. (38), e1793-e1793 (2010).</li><li>Arbab, A. S., Yocum, G. T., Wilson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transfection+agents+in+forming+complexes+with+ferumoxides,+cell+labeling+efficiency,+and+cellular+viability.">Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.</a> Mol Imaging. 3, 24-32 (2004).</li><li>Babic, M., Horak, D., Trchova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(L-lysine)-modified+iron+oxide+nanoparticles+for+stem+cell+labeling.">Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.</a> Bioconjug Chem. 19, 740-750 (2008).</li><li>Golovko, D. M., T, H. e. n. n. i. n. g., Bauer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+stem+cell+labeling+with+ferucarbotran+and+protamine.">Accelerated stem cell labeling with ferucarbotran and protamine.</a> Eur. Radiol. 20, 640-648 (2010).</li><li>Lu, M., Cohen, M. H., Rieves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+report:+Ferumoxytol+for+intravenous+iron+therapy+in+adult+patients+with+chronic+kidney+disease.">FDA report: Ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease.</a> Am. J. Hematol. 85, 315-319 (2010).</li></ol>

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Migliorare l&#39;efficacia di engraftments cellule staminali è fondamentale per il progresso della medicina rigenerativa. Una tecnica non invasiva di visualizzazione per le cellule staminali in vivo migliora in modo significativo la nostra capacità di comprendere i meccanismi che portano a risultati attecchimento di successo. Etichettatura magnetica per la visualizzazione MR, come la procedura abbiamo dimostrato, permette il monitoraggio in vivo di cellule staminali con la RM. Le cellule staminali ma...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> D-MEM glucosio </td><td> Sigma </td><td> D5648 </td><td> O medio altra base per la linea di cellule staminali desiderato da utilizzare </td></tr><tr><td> D-PBS (Ca + +, Mg + + libero) </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td> Tripsina-EDTA 0,05% </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> Siero fetale bovino (FBS) </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol (Feraheme) </td><td> AMAG </td><td> 59338-0775-01 </td><td></td></tr><tr><td> Solfato di protamina </td><td> APP Pharm. </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

Terapie basate sulle cellule staminali offrono un potenziale significativo per il settore della medicina rigenerativa. Tuttavia, molto resta ancora da capire per quanto riguarda la cinetica in vivo di cellule trapiantate. Un metodo non invasivo per monitorare ripetutamente le cellule staminali trapiantate in vivo avrebbe permesso agli investigatori di monitorare direttamente i trapianti di cellule staminali e di individuare gli esiti attecchimento di successo o insuccesso. Una vasta gamma di cellule staminali continua ad essere indagato per innumerevoli applicazioni. Questo protocollo si concentra su tre diverse popolazioni di cellule staminali: embrionali umane del rene 293 (HEK293), le cellule, cellule staminali mesenchimali (hMSC) e staminali pluripotenti indotte (iPS), cellule. HEK 293 cellule derivano da cellule embrionali renali in coltura con tranciati adenovirus 5 del DNA. Queste cellule sono ampiamente utilizzati nella ricerca perché sono facilmente colta, crescono velocemente e sono facilmente transfettate. hMSCs si trovano nel midollo adulti. Queste cellule can essere replicate come cellule indifferenziate, mantenendo multipotenza o la potenzialità di differenziarsi in un numero limitato di destini delle cellule. hMSCs possono differenziare a lignaggi dei tessuti mesenchimali, tra cui gli osteoblasti, adipociti, condrociti, tendini, muscoli e stroma del midollo. le cellule iPS sono geneticamente cellule adulte riprogrammate che sono stati modificati per esprimere geni e fattori simili a definire le proprietà delle cellule staminali embrionali. Queste cellule sono pluripotenti senso che hanno la capacità di differenziarsi in tutte le linee cellulari 1. Sia hMSCs e le cellule iPS hanno dimostrato la capacità di rigenerazione dei tessuti in vivo. Risonanza magnetica (RM) con l&#39;uso di etichette superparamagnetiche nanoparticelle di ossido di ferro (SPIO) delle cellule si sono dimostrati efficaci per il monitoraggio in vivo di cellule staminali a causa della risoluzione anatomica microscopica vicino, un sangue più lunga emivita che permette l&#39;imaging longitudinale e la alto sensitivity per il rilevamento delle cellule forniti da RM di SPIO nanoparticelle 2-4. Inoltre, la RM con l&#39;uso di SPIOs è clinicamente traducibile. SPIOs sono composti da un nucleo di ossido di ferro con uno strato superficiale destrano, carboxydextran o amido che serve per contenere il nocciolo bioreactive ferro da componenti del plasma. Questi agenti creare disomogeneità locali del campo magnetico che portano ad una diminuzione del segnale in immagini T2-pesate RM 5. Purtroppo, SPIOs non vengono più prodotte. Seconda generazione, ultrasmall SPIOs (USPIO), tuttavia, offrono una valida alternativa. Ferumoxytol (FerahemeTM) è uno USPIO composto da un non-stechiometrico magnetite nucleo circondato da un mantello polyglucose carboxymethylether sorbitolo. Il colloidale, dimensione delle particelle di 17-30 nm ferumoxytol è determinato dal dispersione della luce. Il peso molecolare è di 750 kDa, e il relassività costante a campo 2T MRI è 58,609 MM-1 sec-1 forza 4. Ferumoxytol è stato recentemente dalla FDA ha approvato uns un integratore di ferro per il trattamento della carenza di ferro nei pazienti con insufficienza renale 6. Il nostro gruppo ha applicato questo agente in un &quot;off label&quot; l&#39;uso per applicazioni di etichettatura delle cellule. La nostra tecnica dimostra efficace di etichettatura di cellule staminali con ferumoxytol che porta a significativi effetti segnale RM di cellule etichettate sulle immagini MR. Questa tecnica può essere applicata per monitoraggio non invasivo di terapie con cellule staminali in ambito pre-clinico e clinico.

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Abbiamo descritto una tecnica per l&#39;etichettatura e tracciabilità delle cellule staminali con la FDA ha approvato, superparamagnetiche ossido di ferro (SPIO), ferumoxytol (Feraheme). Questa tecnica di imaging cellulare che utilizza la risonanza magnetica (RM) per la visualizzazione, è facilmente accessibile per monitoraggio a lungo termine e la diagnosi di engraftments staminali successo o insuccesso delle cellule nei pazienti.

Etichettatura cellule staminali con Ferumoxytol, un approvati dalla FDA nanoparticelle di ossido di ferro

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

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Medicine

20.7K Views.  Molecular Imaging Program at Stanford (MIPS). Abbiamo descritto una tecnica per l'etichettatura e tracciabilità delle cellule staminali con la FDA ha approvato, superparamagnetiche ossido di ferro (SPIO), ferumoxytol (Feraheme). Questa tecnica di imaging cellulare che utilizza la risonanza magnetica (RM) per la visualizzazione, è facilmente accessibile per monitoraggio a lungo termine e la diagnosi di engraftments staminali successo o insuccesso delle cellule nei pazienti.

Video: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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Medicina

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Co-coltura di sistema con un Organotipica cervello Slice e 3D Spheroid delle cellule di carcinoma

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

Continua trasfusione di cambio manuale per i pazienti con anemia falciforme: un metodo efficace per evitare sovraccarico di ferro

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Sintesi, caratterizzazione e applicazione di nanosondanti di ossido di ferro superparamagnetico per il rilevamento della tubercolosi extrapolmonare

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Nanoparticelle di ossido di ferro rivestite con Polyethyleneimine come un veicolo per la consegna di piccolo RNA interferente ai macrofagi In Vitro e In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Un metodo privo di enzimi per l'isolamento e l'espansione delle cellule staminali mesenchimale derivate dall'Adipo

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Monitoraggio delle cellule staminali mesenchymale con etichetta chimica dell'ossido di ferro superparamagnetico utilizzando la risonanza magnetica dopo la consegna intranasale in un modello murino traumatico da lesioni cerebrali

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Stima dei nanocristalli urinari nell'uomo utilizzando l'etichettatura del fluoroforo di calcio e l'analisi di tracciamento delle nanoparticelle

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...