Si ringrazia il Dr. Leila Shakeri &amp; Dr. Aref Dournaei per la loro discus critica e Mohammad Reza Khadem Sharif per i suoi supporti tecnici.

1. Preparare la soluzione enzimatica e medie proliferazione (PM) <ol><li> Marca Collagenasi I Soluzione: Pesare, collagenasi di tipo I (12 mg / ml) e sciogliere in 1 ml di PBS e filtro con un filtro 0,2 micron siringa. Poi posto 15 ml tubetto e la tiene a -20 ° C fino al momento dell&#39;uso. </li><li> Fai Soluzione dispasi: Pesare dispasi (16 mg / ml) e sciogliere in 1 ml di PBS e filtro con un filtro 0,2 micron siringa. Poi posto 15 ml tubetto e la tiene a 4 ° C fino al momento dell&#39;uso. </li><li> Rendere soluzione enzimatica: Aggiungere 1 ml di collagenasi di tipo I soluzioni (12 mg / ml) e 1 ml di soluzioni dispasi (16 mg / ml) in 2 ml di PBS sterile contenente 100 mg / ml di penicillina, 100 mg / ml di streptomicina. Concentrazione totale di dispasi e collagenasi I in volume finale deve essere di 4 mg / ml e 3 mg / ml, ricettivamente. Quindi, in questo volume aliquota di quattro tubo 15 ml, ciascuna contenente 1 ml di soluzione di enzima. Ogni tubo potrebbe essere utilizzato per una digestione pasta. </li><li> Rendere soluzione di lavaggio: Aggiungere 100 mg / ml di penicillina, 100 mg / ml di streptomicina in PBS. </li><li> Assicurarsi che i supporti di base: Alpha modifica del mezzo di Eagle (α-MEM) addizionato con 10% FBS e 100 unità / ml di penicillina, 100 mg / ml di streptomicina. </li><li> Rendere il supporto proliferazione (PM): modifica Alpha di mezzo di Eagle (α-MEM) addizionato con 10% FBS, 100 mM L-ascorbico 2-fosfato, 2 mM L-glutammina, 100 unità / ml di penicillina, 100 mg / ml streptomicina, 0,25 mg / ml di amfotericina B. </li><li> Rendere supporti odontogene: modifica Alpha di mezzo di Eagle (α-MEM) addizionato con 10% FBS, 100 mM L-ascorbico 2-fosfato, 2 mM L-glutammina, 100 unità / ml di penicillina, 100 mg / ml di streptomicina, 0,01 pM desametasone, 5 mM β-glicerolo fosfato, 1,8 mM di fosfato monopotassico. </li></ol> 2. Preparare umano tessuto dentale Dental Pulp per Isolatio cellule staminali della polpan <ol><li> Normali molari umani terzi sono stati raccolti da adulti (21-29 anni di età) presso la Clinica Odontoiatrica dell&#39;Imam Ali sotto Institutional Review Board approvato (IRB). </li><li> Denti sono stati inseriti nel terreno base (α-MEM supplementato con 10% FBS) e sono stati trasferiti in laboratorio a 4 ° C. </li><li> Sotto la condizione sterile, lavorando all&#39;interno di una cappa a flusso laminare biohazard, set-up è stato fatto uno 100 millimetri Petri per ogni dente da trattare. Superfici dentali venivano pulite del 70% di etanolo. </li><li> Mentre lavora in uno dei piatti Petri, dente è stato tenuto da pinze dentali e una lama di bisturi è stato usato per pulire i detriti e legamento parodontale sulle radici. </li><li> Giunzione smalto-cemento è stato lentamente tagliato utilizzando sterilizzato disco dentale per rivelare la camera pulpare. Va considerato che il processo di taglio deve essere effettuata lentamente per ridurre il surriscaldamento del tessuto dentale. </li><li> Tessuto pulpare è stato delicatamente separato dalla corona e radice. </li&gt; <li> Tessuto pulpare è stato tagliato in piccoli pezzi con l&#39;aiuto di una lama di bisturi. </li></ol> 3. Umano Isolamento polpa dentale <ol><li> Pulp tessuti sono stati sottoposti sia dissociazione enzimatica del tessuto della polpa o cellule conseguenza diretta da espianti pasta tessuto per l&#39;isolamento di hDPSCs. </li><li> Dissociazione enzimatica del tessuto della polpa: <ol><li> Piccoli pezzi di tessuti paste sono state trasferite in soluzione enzimatica per 1 ora a 37 ° C. Vortex ogni 30 minuti per rompere il tessuto. </li><li> In seguito, gli aggregati a grandi cellule sono state rimosse e cella singola sospensioni sono stati ottenuti facendo passare le cellule attraverso un 70-mM filtro cella. </li><li> Monocellulari sospensioni sono state centrifugate a 1200 rpm per 5 min a temperatura ambiente. </li><li> Surnatanti sono stati accuratamente pipettati off e pellet è stato risospeso in 1 ml di terreno di proliferazione (PM). (Nota: FBS a medio proliferazione terminare enzimatica disciazione.) </li><li> Sospensioni monocellulari di polpa dentale è stato seminato in 25 cm 2 pallone di coltura con PM e poi incubate a 37 ° C in 5% CO 2. </li><li> Il terreno di coltura è stato cambiato ogni tre giorni fino a quando la confluenza delle cellule è stato raggiunto. </li></ol></li><li> Cella diretta conseguenza da espianti di tessuto pasta: <ol><li> Tessuto pulpare stata macinata in 1-2-millimetri frammenti, e ogni pezzo è stato posto in 25-cm 2 fiasche con PM e poi incubate a 37 ° C in 5% CO 2. Si deve considerare che il volume totale del PM per conseguenza di cella deve supportare il fissaggio di pezzi paste per outgrowth cella ulteriore (2-3 ml per beuta). </li><li> Medio è stato modificato dopo escrescenza è stata osservata. </li><li> Le cellule erano cresciuti a confluenza sub-coltura con un rapporto di 1:4 (passaggio 1). </li></ol></li></ol> 4. Immunofenotipizzazione <ol><li> 250.000 cellule /tubo di entrambi i tipi di cellule nel passaggio 3 sono state incubate da PE o FITC-coniugato anti-umani coniugati con i loro isotipi per 30 min a 4 ° C al buio. (Nota:. Concentrazione di anticorpi è stato applicato secondo il protocollo fabbricazione) </li><li> Dopo il tempo di incubazione, 100 microlitri di PBS o soluzione di diluizione FACS è stato aggiunto a ciascuna provetta e centrifugati a 1200 rpm per 5 min in luogo buio. </li><li> Surnatanti sono stati rimossi e pellet sono stati risospesi in 100 pl di PBS o diluizione FACS in luogo buio. </li><li> Marcatori di superficie mirate (marcatori CD) sono stati valutati da BD FACSCalibur. </li></ol> 5. Induzione di differenziazione delle cellule in DPSCs mineralizzati e quantificata Assay Rosso alizarina S <ol><li> DPSCs che sono stati ottenuti da una dissociazione enzimatica del tessuto pulpare (noto come DPSC-DE) o conseguenza delle cellule direttamente da espianti di tessuto polpa (DPSC-OG) sono stati sub-coltivate per 3 passaggi. </li><li> Lat Passage 3, le cellule sono state seme in una piastra a 6 pozzetti 5000 cellule / pozzetto. </li><li> Alla confluenza del 60%, medie odontogeno stati sostituiti da PM convenzionale. Tre pozzetti erano rimasti come controllo negativo aggiungendo mezzo di crescita. </li><li> Media ogni 3 giorni fino al giorno 21. </li><li> Al giorno 21, cellule sono state lavate con PBS e sono stati fissati con 1 ml / pozzetto 10% paraformaldeide per 15 min a temperatura ambiente. </li><li> Poi paraformaldeide stato pipettato off, cura e le cellule sono state lavate 3 volte (5-10 minuti per ogni volta) con acqua distillata H 2 O. (Nota: evitare di disturbare il monostrato.) </li><li> Dopo il lavaggio, 1 ml / pozzetto di rosso alizarina S sono stati aggiunti per 20 minuti a temperatura ambiente. </li><li> Le cellule sono state lavate da H 2 O deionizzata quattro volte (5 min per ogni volta con gentile dondolio) per rimuovere qualsiasi ulteriore Alizarin rosso. </li><li> 1 ml / pozzetto H 2 O sono stati aggiunti per impedire alle cellule di essiccazione. </li><li> Le piastre sono stati assunti i componenti visivipection e / o l&#39;acquisizione dell&#39;immagine. </li><li> Per il saggio di quantificazione Rosso alizarina S, H 2 O sono stati sostituiti da 1 ml di acido acetico 10% in ciascun pozzetto della piastra da 6 pozzetti e incubare per 30 min a temperatura ambiente con agitazione. </li><li> Poi, monostrato di cellule / pozzetto sono stati raschiare e sia l&#39;acido acetico e le cellule sono state trasferite nelle distinte da 1,5 ml micro-centrifuga tubi. (Nota:. Ogni bene dovrebbe essere il trasferimento in un tubo separato vale a dire tre pozzi di prova e tre pozzi di controllo per ogni gruppo ED &amp; OG) </li><li> Tubi furono vortexate energicamente per 30 sec. </li><li> Riscaldare a 85 ° C per 10 min. (Nota: per evitare l&#39;evaporazione, i tubi sono stati sigillati con parafilm.) </li><li> Dopo il riscaldamento, i tubi sono stati trasferiti in ghiaccio per 5 min. (Nota: I tubi non devono essere aperti finché diventano raffreddato.) </li><li> Gli impasti sono stati centrifugati a 20.000 xg per 15 min. Mentre centrifugazione, alizarina standard Red sono stati effettuatiup. </li></ol> Standard Rosso alizarina S sono state preparate prima diluendo il tampone di diluizione e usato per fare diluizioni seriali due volte in base alla tabella. <table border="1"><tbody><tr><td colspan="7"> Gamma Alta concentrazione di colorante </td><td colspan="7"> Gamma bassa concentrazione di colorante </td></tr><tr><td> 2 mM </td><td> 1 mM </td><td> 500 pM </td><td> 250 pM </td><td> 125 pM </td><td> 62,5 pM </td><td> 31,3 pM </td><td> 15,6 pM </td><td> 7,8 pM </td><td> 3,9 pM </td><td> 1,9 pM </td><td> 0,9 pM </td><td> 0,4 micron </td><td> Vuoto </td></tr></tbody></table><ol start="16"><li> Dopo la centrifugazione, 1 ml di surnatante / tubo sono state trasferite nei nuovi separati 1,5 ml micro-provette da centrifuga. </li><li> Il pH è stato neutralizzato con ~ 375 microlitri 10% Idrossido di ammonio per provetta. (Nota: la rabbia pH dovrebbe essere 4,1-4,5.) </li><li> 150 ml di standard e campioni (include test separati e controlli) sono stati aggiunti in una parete opaca, fondo trasparente piastra a 96 pozzetti. </li><li> L&#39;assorbanza è stata letta a 405 nm e valori di assorbanza dei campioni sono stati confrontati con trama standard per ulteriori analisi. </li></ol>

<ol>
	<li>Volponi, A. A., Pang, Y., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+biological+tooth+repair+and+regeneration.">Stem cell-based biological tooth repair and regeneration.</a> Trends Cell Biol. 20, 715-722 (2010).</li><li>Nosrat, I. V., Widenfalk, J., Olson, L., Nosrat, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+cells+produce+neurotrophic+factors,+interact+with+trigeminal+neurons+in+vitro,+and+rescue+motoneurons+after+spinal+cord+injury.">Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury.</a> Dev. Biol. 238, 120-132 (2001).</li><li>Gandia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells+improve+left+ventricular+function,+induce+angiogenesis,+and+reduce+infarct+size+in+rats+with+acute+myocardial+infarction.">Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.</a> Stem Cells. 26, 638-645 (2008).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proc. Natl. Acad. Sci. U. S. A.. 97, 13625-13630 (2000).</li><li>Graziano, A., d'Aquino, R., Laino, G., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells:+a+promising+tool+for+bone+regeneration.">Dental pulp stem cells: a promising tool for bone regeneration.</a> Stem Cell Rev. 4, 21-26 (2008).</li><li>Kerkis, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+transplantation+of+human+immature+dental+pulp+stem+cells+from+baby+teeth+to+golden+retriever+muscular+dystrophy+(GRMD)+dogs:+Local+or+systemic.">Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic.</a> J. Transl. Med. 6, 35 (2008).</li><li>Onyekwelu, O., Seppala, M., Zoupa, M., Cobourne, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+development:+2.+Regenerating+teeth+in+the+laboratory.">Tooth development: 2. Regenerating teeth in the laboratory.</a> Dent. Update. 34, 20-29 (2007).</li><li>Cordeiro, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+tissue+engineering+with+stem+cells+from+exfoliated+deciduous+teeth.">Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth.</a> J. Endod. 34 (08), 962-969 (2008).</li><li>Pierdomenico, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+mesenchymal+stem+cells+with+immunosuppressive+activity+can+be+easily+isolated+from+dental+pulp.">Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp.</a> Transplantation. 80, 836-842 (2005).</li><li>de Mendonca Costa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+large+cranial+defects+in+nonimmunosuppressed+experimental+design+with+human+dental+pulp+stem+cells.">Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells.</a> J. Craniofac. Surg. 19, 204-210 (2008).</li><li>Tirino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+identification,+characterization+and+banking+of+human+DPSCs:+current+strategies+and+perspectives.">Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives.</a> Stem Cell Rev. 7, 608-615 (2011).</li><li>Tirino, V., Paino, F., De Rosa, A., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+characterization,+and+banking+of+human+dental+pulp+stem+cells.">Identification, isolation, characterization, and banking of human dental pulp stem cells.</a> Methods Mol. Biol. 879, 443-463 (2012).</li><li>Eslaminejad, M. B., Vahabi, S., Shariati, M., Nazarian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Growth+and+Characterization+of+Stem+Cells+from+Human+Dental+Pulp+of+Deciduous+Versus+Permanent+Teeth.">In vitro Growth and Characterization of Stem Cells from Human Dental Pulp of Deciduous Versus Permanent Teeth.</a> J. Dent. (Tehran). 7, 185-195 (2010).</li><li>Wei, X., Ling, J., Wu, L., Liu, L., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mineralization+markers+in+dental+pulp+cells.">Expression of mineralization markers in dental pulp cells.</a> J. Endod. 33, 703-708 (2007).</li><li>Nombela-Arrieta, C., Ritz, J., Silberstein, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elusive+nature+and+function+of+mesenchymal+stem+cells.">The elusive nature and function of mesenchymal stem cells.</a> Nat. Rev. Mol Cell Biol. 12, 126-131 (2011).</li><li>Huang, G. T., Gronthos, S., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+dental+tissues+vs.+those+from+other+sources:+their+biology+and+role+in+regenerative+medicine.">Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.</a> J. Dent. Res. 88, 792-806 (2009).</li><li>Graziano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold's+surface+geometry+significantly+affects+human+stem+cell+bone+tissue+engineering.">Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.</a> J. Cell Physiol. 214, 166-172 (2008).</li><li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells:+from+biology+to+clinical+applications.">Human dental pulp stem cells: from biology to clinical applications.</a> J. Exp. Zool. B. Mol. Dev. Evol. 312, 408-415 (2009).</li><li>Mitsiadis, T. A., Feki, A., Papaccio, G., Caton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells,+niches,+and+notch+signaling+in+tooth+injury.">Dental pulp stem cells, niches, and notch signaling in tooth injury.</a> Adv. Dent. Res. 23, 275-279 (2011).</li><li>Shi, S., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp.">Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp.</a> J. Bone Miner. Res. 18, 696-704 (2003).</li><li>Tomic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+properties+of+mesenchymal+stem+cells+derived+from+dental+pulp+and+dental+follicle+are+susceptible+to+activation+by+toll-like+receptor+agonists.">Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.</a> Stem Cells Dev. 20, 695-708 (2011).</li><li>Tsukamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mineralized+nodule+formation+by+cultures+of+human+dental+pulp-derived+fibroblasts.">Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.</a> Arch. Oral Biol. 37, 1045-1055 (1992).</li><li>About, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dentin+production+in+vitro.">Human dentin production in vitro.</a> Exp. Cell Res. 258, 33-41 (2000).</li><li>Couble, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odontoblast+differentiation+of+human+dental+pulp+cells+in+explant+cultures.">Odontoblast differentiation of human dental pulp cells in explant cultures.</a> Calcif. Tissue Int. 66, 129-138 (2000).</li><li>Onishi, T., Kinoshita, S., Shintani, S., Sobue, S., Ooshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+proliferation+and+differentiation+of+dog+dental+pulp+cells+in+serum-free+culture+medium+by+insulin-like+growth+factor.">Stimulation of proliferation and differentiation of dog dental pulp cells in serum-free culture medium by insulin-like growth factor.</a> Arch. Oral Biol. 44 (99), 361-371 (1999).</li><li>Huang, G. T., Sonoyama, W., Chen, J., Park, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+human+dental+pulp+cells:+various+isolation+methods+and+culturing+environments.">In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.</a> Cell Tissue Res. 324, 225-236 (2006).</li><li>Bakopoulou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo/odontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth.">Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.</a> Calcif. Tissue Int. 88, 130-141 (2011).</li><li>Curtis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells.">EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.</a> BMC Mol. Biol. 11, 61 (2010).</li><li>Suchanek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells+and+their+characterization.">Dental pulp stem cells and their characterization.</a> Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li><li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li><li>Atari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

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Questo protocollo descrive il processo di isolamento e caratterizzazione di hDPSCs da polpa dentale utilizzando due metodi, dissociazione enzimatica e la conseguenza diretta di cellule staminali da espianti di tessuto pulpare. Inoltre, in vitro differenziazione di queste cellule in odontoblasti, è stata valutata mediante dosaggio quantitativo Rosso alizarina S &amp; QPCR. Protocolli esistenti per isolare dal tessuto pulpare dente umano era stato utilizzato diversi strumenti quali p...

Le cellule staminali sono cellule clonogeniche che possiedono due caratteristiche notevoli, conosciuti come multi-potenza e di auto-rinnovamento 15. Tra tutte le cellule staminali con diverse potenze replicative, le cellule staminali dentali come le cellule staminali post-natali hanno attirato l&#39;attenzione negli ultimi anni, a causa della loro accessibilità, plasticità, e ad alta capacità proliferativa rispetto ad altre cellule staminali adulte 16. Caratteristicamente, simili alle cellule staminali mesenchimali, cellule staminali polpa dentale sono aderenti cellule clonogeniche aventi capacità multipla differenziazione in mesenchima e / o non-mesenchima lignaggi, sia in vitro che in vivo. 1-8,17,18 DPSCs sono identificati da la loro espressione negativa di antigeni ematopoietiche (ad esempio, CD45, CD34, CD14), e l&#39;espressione di CD90 positivo, CD29, CD73, CD105, CD44 e STRO-1. 19,20 Facile potenziale ottenuto con un minimo sforzo e la morbilità rendere DPSCs umani come un validosorgente in grado di MSC rispetto alle fonti ordinarie, come le cellule staminali mesenchimali del midollo 21. In generale, DPSCs sono stati isolati da una escrescenza metodo, cioè, la migrazione di cellule staminali da espianti di tessuto pulpare (DPSC-OG) 22-24, e / o mediante digestione enzimatica (DPSC-ED) 4,6,25. Studi precedenti hanno dimostrato che il metodo di isolamento e condizioni di coltura possono indurre differenti popolazioni o casate sotto passaggi in vitro 26,27. Nel caso di denti permanenti (pDPSCs), Huang et al rivelato che enzimatiche pDPSCs digeriti hanno potenziale proliferativo superiore rispetto ai DPSCs cresciuti. 26 Inoltre, nel caso di denti decidui (dDPSCs), è stato dimostrato che STRO-1 e CD34 marcatori espressi più dDPSC-DE rispetto a dDPSC-OG. Inoltre, dDPSC-ED visualizzato elevato tasso di mineralizzazione in definito osteo / odonto media. 27 Pertanto, a causa del potenziale eccezionale di DPSCs in rmedicina egenerative, ulteriori studi saranno necessari per una migliore comprensione delle possibili varie popolazioni che sono derivati ​​da diversi metodi di isolamento. Qui, era tentativo di introdurre modo semplice di estrazione polpa, utilizzando uno step-disco diamantato dentale per facilitare il processo di estrazione polpa. Inoltre, dopo l&#39;isolamento di polpa umani cellule staminali derivate mediante l&#39;applicazione di metodi di ED o OG, proprietà generali e capacità di differenziazione tra i due gruppi sono stati studiati.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Catalogo o Lotto. numero </td><td> Commenti (opzionale) </td></tr><tr><td> α-MEM </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td> Collagenasi tipo I </td><td> Sigma-Aldrich </td><td> C0130-100MG </td><td></td></tr><tr><td> Dispasi </td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td> Penicillina / streptomicina </td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td> Amfotericina B </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td> Siero fetale bovino definito (FBS) </td><td> HyClone </td><td> SH30070.03 </td><td></td></tr><tr><td> L-ascorbico 2-fosfato </td><td> Sigma </td><td> A8960-5G </td><td></td></tr><tr><td> L-glutammina </td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td> Desametasone </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> β-Glicerolo sale idrato fosfato disodico, BioUltra </td><td> Sigma </td><td> G9422-100G </td><td></td></tr><tr><td> Potassio fosfato monobasico </td><td> Sigma-Aldrich </td><td> P5655 </td><td></td></tr><tr><td> Osteogenesi Assay Kit </td><td> Millipore </td><td> PS01802031 </td><td></td></tr><tr><td> Mouse IgG2b-PE isotipo di controllo </td><td> BD Pharmingen </td><td> 50808088029 </td><td></td></tr><tr><td> FITC del mouse IgG2b isotipo di controllo </td><td> BD Pharmingen </td><td> 559532 </td><td></td></tr><tr><td> Del mouse FITC IgG1 isotipo κ </td><td> BD Pharmingen </td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE topo anti-umano CD34/CD45 </td><td> BD Pharmingen </td><td>341071 </td><td></td></tr><tr><td> PE anti-CD146 umano </td><td> BD Pharmingen </td><td> 550315 </td><td></td></tr><tr><td> Monoclonale murino anti-umano CD90/FITC </td><td> Daka </td><td> 00034418 </td><td></td></tr><tr><td> PE di topo anti-CD73 umano </td><td> BD Pharmingen </td><td> 550257 </td><td></td></tr><tr><td> Anti-h CD105/Endoglin PE </td><td> BD Pharmingen </td><td> FAB10971P </td><td></td></tr><tr><td> PE topo anti-umana CD11b/Mac1 </td><td> BD Pharmingen </td><td> 5553888 </td><td></td></tr><tr><td> CD44 PE topo anti umano </td><td> BD Pharmingen </td><td> 555479 </td><td></td></tr><tr><td> Soluzione tampone fosfato (PBS) </td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70-micron cella filtro </td><td> Falco </td><td> 352360 </td><td></td></tr><tr><td&gt; 0,2 micron filtro a siringa </td><td> Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25 cm 2 cultura pallone </td><td> Sigma-Aldrich </td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> ATTREZZATURE </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342975 </td><td></td></tr><tr><td> Multiskan spettrofotometro per micropiastre </td><td> Thermo Scientific </td><td> 51119200 </td><td></td></tr><tr><td> Fleurcense Microscopio </td><td> Olimpo </td><td></td><td></td></tr><tr><td> Flowing Versione software 2.3.1 </td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"&gt; Denti del giudizio in via di sviluppo sono facilmente accessibile fonte di cellule staminali durante l&#39;età adulta che potrebbe essere ottenuta con trattamenti ortodontici di routine. Umani polpa cellule staminali derivate (hDPSCs) in possesso di un elevato potenziale di proliferazione con il multi-lignaggio capacità di differenziazione confrontare alla fonte ordinaria di cellule staminali adulte<sup&gt; 1-8</sup&gt;, Quindi, hDPSCs potrebbero essere i candidati ideali per il trapianto autologo in ingegneria dei tessuti e medicina rigenerativa. Oltre a questi benefici, in possesso delle cellule staminali mesenchimali (MSC) offre, come effetti immunolodulatory, hDPSCs rendere più prezioso, anche nel caso di trapianto allogenico<sup&gt; 6,9,10</sup&gt;. Pertanto, il passaggio principale per utilizzare questa fonte di cellule staminali è quello di selezionare il miglior protocollo per isolare hDPSCs dal tessuto pulpare. Al fine di raggiungere questo obiettivo, è fondamentale per studiare l&#39;effetto delle condizioni di isolamento diverse su differenti comportamenti cellulari, come i loro marcatori di superficie comuni e anche la loro capacità di differenziazione.</p&gt;<p class="jove_content"&gt; In questo modo, qui si separano tessuti umani colpiti dalla polpa terzi denti molari, e poi usato entrambi i protocolli esistenti basati sulla letteratura, per hDPSCs isolamento,<sup&gt; 11-13</sup&gt;<em&gt; Per esempio</em&gt; Dissociazione enzimatica del tessuto pulpare (DPSC-DE) o escrescenza da espianti di tessuto (DPSC-OG). A questo proposito, abbiamo cercato di facilitare i metodi di isolamento mediante disco diamantato dentale. Quindi, queste cellule caratterizzato in termini di stromale associati marcatori (CD73, CD90, CD105 e CD44), pennarelli emopoietiche / endoteliale (CD34, CD45 e CD11b), marcatori perivascolare, come CD146 e anche STRO-1. Successivamente, questi due protocolli sono stati confrontati in base alla potenza differenziazione in odontoblasti sia quantitativa reazione a catena della polimerasi (QPCR) e Alizarin colorazione rossa. QPCR sono stati utilizzati per valutare l&#39;espressione dei geni correlati mineralizzazione (fosfatasi alcalina; ALP, phosphoglycoprotein matrice extracellulare; MEPE &amp; dentina sialophosphoprotein; DSPP).<sup&gt; 14</sup</p

<ol><li> DPSCs che sono stati ottenuti dalla dissociazione enzimatica (DPSC-ED) sono mostrati qui a 10 giorni, 15,18 (Figura 1). Le colonie avviato per formare su quasi 3 a 5 giorni dopo l&#39;isolamento. </li><li> DPSCs superato (DPSC-OG) sono mostrati nella Figura 2, il giorno 5, 10, 13 e 18. Fibroblasto-simili cellule hanno cominciato a migrare dal tessuto pulpare nel pallone da parallelo ordinazione quasi 5 giorni dopo la semina. </li><li> DPSCs al passaggio 3 utilizzando entrambi i metodi sono illu...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

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

Il metodo descritto l&#39;isolamento e la caratterizzazione di cellule staminali dentali umane Pulp (hDPSCs) utilizzando<strong&gt; Dissociazione enzimatica della polpa (DPSC-DE)</strong&gt; O<strong&gt; Conseguenza diretta di cellule staminali da espianti di tessuto pulpare (DPSC-OG). Seguito da</strong&gt;<em&gt; In vitro</em&gt; Differenziazione comparativa di entrambi i tipi di hDPSCs in odontoblasti.

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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Research

JoVE Journal

Medicine

26.2K Views.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Il metodo descritto l'isolamento e la caratterizzazione di cellule staminali dentali umane Pulp (hDPSCs) utilizzando Dissociazione enzimatica della polpa (DPSC-DE) O Conseguenza diretta di cellule staminali da espianti di tessuto pulpare (DPSC-OG). Seguito da In vitro Differenziazione comparativa di entrambi i tipi di hDPSCs in odontoblasti.

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

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Ricerca

Medicina

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caratterizzazione funzionale di Human ventricolare Cardiomyocytes da campioni freschi chirurgici

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Due metodi per stabilire primarie cellule umane endometriale stromali da isterectomia campioni

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Isolamento e Cellular Fenotipizzazione di cellule staminali mesenchimali derivate dal liquido sinoviale e del midollo osseo di mini-pig

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caratterizzazione di cellule immunitarie nel tessuto adiposo umano mediante citometria a flusso

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Formazione di Biofilm orali su differenti materiali per impianti dentali

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

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. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Ex Vivo espansione di cellule staminali da cordone ombelicale umano derivate dal sangue CD34+ cellule utilizzando acido valproico

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caratterizzazione citometrica a flusso simultaneo di più tipi di cellule recuperate dal cervello del mouse/cavo spinale attraverso diversi metodi di omogeneizzazione

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...