Name: isolation, culture, and transplantation of muscle satellite cells
Uploaded: 2014-04-08T18:00:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Ringraziamo il Dott. Shahragim Tajbakhsh per la fornitura di topi Myf5 + / nLacZ. Ringraziamo anche Alexander Hron e Michael Baumrucker per la lettura critica di questo manoscritto. Questo lavoro è stato sostenuto da finanziamenti della Muscular Dystrophy Association (MDA) e Gregory Marzolf Jr. MD Centro Award.

Gli animali sono stati alloggiati in un ambiente SPF e sono stati monitorati dalla Direzione Risorse ricerca su animali (RAR) dell&#39;Università del Minnesota. . Gli animali sono stati sacrificati con mezzi adeguati (CO 2 inalazione o iniezione KCl dopo essere stati anestetizzati con iniezione IP di Avertin (250 mg / kg) Tutti i protocolli sono stati approvati dalla cura degli animali ed uso Comitato Istituzionale (IACUC, numero di codice: 1304-30.492 ) dell&#39;Università del Minnesota. <p class="jove...

<ol>
	<li>Fukada, 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=Purification+and+cell-surface+marker+characterization+of+quiescent+satellite+cells+from+murine+skeletal+muscle+by+a+novel+monoclonal+antibody.">Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody.</a> Exp. Cell Res. 296, 245-255 (2004).</li><li>Hirai, H., Verma, M., Watanabe, S. C. T., Asakura, Y., Asakura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD+regulates+apoptosis+of+myoblasts+through+microRNA-mediated+down-regulation+of+Pax3.">MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3.</a> J. Cell Biol. (191), 347-365 (2010).</li><li>Asakura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+adult+skeletal+muscle.">Stem cells in adult skeletal muscle.</a> Trends Cardiovasc. Med. 13, 123-128 (2003).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+that+participate+in+regeneration+of+skeletal+muscle.">Cells that participate in regeneration of skeletal muscle.</a> Gene Ther. 9, 752-753 (2002).</li><li>Collins, C. 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=Stem+cell+function,+self-renewal,+and+behavioral+heterogeneity+of+cells+from+the+adult+muscle+satellite+cell+niche.">Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.</a> Cell. 122, 289-301 (2005).</li><li>Montarras, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. 456, 502-506 (2008).</li><li>Conboy, M. J., Cerletti, M., Wagers, A. J., Conboy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-analysis+and+FACS+sorting+of+adult+muscle+fiber-associated+stem/precursor+cells.">Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells.</a> Methods Mol. Biol. 621, 165-173 (2010).</li><li>Yokoyama, T., Huard, J., Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+therapy+for+stress+urinary+incontinence+and+bladder+dysfunction.">Myoblast therapy for stress urinary incontinence and bladder dysfunction.</a> World J. Urol. 18, 56-61 (2000).</li><li>Menasche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+satellite+cell+transplantation.">Skeletal muscle satellite cell transplantation.</a> Cardiovasc. Res. 58, 351-357 (2000).</li><li>Huard, 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=Myoblast+transplantation+produced+dystrophin-positive+muscle+fibres+in+a+16-year-old+patient+with+Duchenne+muscular+dystrophy.">Myoblast transplantation produced dystrophin-positive muscle fibres in a 16-year-old patient with Duchenne muscular dystrophy.</a> Clin. Sci. 81, 287-288 (1991).</li><li>Tremblay, J. P., 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=Results+of+a+triple+blind+clinical+study+of+myoblast+transplantations+without+immunosuppressive+treatment+in+young+boys+with+Duchenne+muscular+dystrophy.">Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy.</a> Cell Transplant. 2, 99-112 (1993).</li><li>Gussoni, E., Blau, H. M., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+individual+myoblasts+after+transplantation+into+muscles+of+DMD+patients.">The fate of individual myoblasts after transplantation into muscles of DMD patients.</a> Nat. Med. 3, 970-977 (1997).</li><li>Palmieri, B., Tremblay, J. P., Daniele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past,+present+and+future+of+myoblast+transplantation+in+the+treatment+of+Duchenne+muscular+dystrophy.">Past, present and future of myoblast transplantation in the treatment of Duchenne muscular dystrophy.</a> Pediatr. Transplant. 14, 813-819 (2010).</li><li>Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hydrodynamic+forces+and+apoptosis:+a+complex+question.">Acute hydrodynamic forces and apoptosis: a complex question.</a> Biotechnol. Bioeng. 98, 772-788 (2007).</li><li>Asakura, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+cells+from+diverse+adult+tissues+are+capable+of+in+vitro+hematopoietic+differentiation.">Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation.</a> Exp. Hematol. 30, 1339-1345 (2002).</li><li>Asakura, 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=Increased+survival+of+muscle+stem+cells+lacking+the+MyoD+gene+after+transplantation+into+regenerating+skeletal+muscle.">Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 16552-16557 (2007).</li><li>Gerard, X., 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=Real-time+monitoring+of+cell+transplantation+in+mouse+dystrophic+muscles+by+a+secreted+alkaline+phosphatase+reporter+gene.">Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.</a> Gene Ther. 16, 815-819 .</li><li>Tajbakhsh, S., Rocancourt, D., Cossu, G., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+genetic+hierarchies+controlling+skeletal+myogenesis+Pax-3+and+Myf-5+act+upstream+of+MyoD.">Redefining the genetic hierarchies controlling skeletal myogenesis Pax-3 and Myf-5 act upstream of MyoD.</a> Cell. 89, 127-138 (1997).</li><li>Beauchamp, J. 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=Expression+of+CD34+and+Myf5+defines+the+majority+of+quiescent+adult+skeletal+muscle+satellite+cells.">Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.</a> J. Cell Biol. 151, 1221-1134 (2000).</li><li>Sabourin, L. A., Girgis-Gabardo, A., Seale, P., Asakura, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+differentiation+potential+of+primary+MyoD-/-+myogenic+cells+derived+from+adult+skeletal+muscle.">Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle.</a> J. Cell Biol. 144, 631-643 (1999).</li><li>Bischoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+single+skeletal+muscle+fibers+in+vitro.">Regeneration of single skeletal muscle fibers in vitro.</a> Anat. Rec. 182, 215-235 (1975).</li><li>Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J. Cell Biol. 159, 123-134 (2002).</li><li>Kuang, S., Kuroda, K., Le Grand, F., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+self-renewal+and+commitment+of+satellite+stem+cells+in+muscle.">Asymmetric self-renewal and commitment of satellite stem cells in muscle.</a> Cell. 129, 999-1010 (2007).</li><li>Cerletti, 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=Highly+efficient,+functional+engraftment+of+skeletal+muscle+stem+cells+in+dystrophic+muscles.">Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.</a> Cell. 134, 37-47 (2008).</li><li>Farina, N. H., 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=A+role+for+RNA+post-transcriptional+regulation+in+satellite+cell+activation.">A role for RNA post-transcriptional regulation in satellite cell activation.</a> Skelet. Muscle. 2, 21-21 (2012).</li><li>Tanaka, K. K., Hall, J. K., Troy, A. A., Cornelison, D. D., Majka, S. M., Olwin, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndecan-4-expressing+muscle+progenitor+cells+in+the+SP+engraft+as+satellite+cells+during+muscle+regeneration.">Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration.</a> Cell Stem Cell. 4, 217-225 (2009).</li><li>Pallafacchina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adult+tissue-specific+stem+cell+in+its+niche:+a+gene+profiling+analysis+of+in+vivo+quiescent+and+activated+muscle+satellite+cells.">An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells.</a> Stem Cell Res. 4, 77-91 (2009).</li></ol>

In questo protocollo, cellule satelliti quiescenti possono essere facilmente purificati da adulto muscolo scheletrico di topi da digestione con collagenasi e separazione MACS mediata da anticorpi superficie. Questo metodo richiede circa 6 ore e non necessita di apparecchiature costose come una macchina FACS. Inoltre, questo metodo è relativamente poco costoso rispetto alla superficie di separazione FACS mediata da anticorpi. Una maggiore resa di cellule satelliti quiescenti si aspetta anche in confronto a FACS per ques...

Cellule satelliti muscolari sono una piccola frazione di cellule staminali miogeniche situati sotto la lamina basale delle fibre muscolari scheletriche. Essi sono caratterizzati dall&#39;espressione di Pax7, Pax3, c-Met, M-caderina, CD34, Syndecan-3, e calcitonina 1 - 3. Le cellule satelliti hanno dimostrato di essere responsabile della rigenerazione muscolare come cellule staminali muscolari. Nel muscolo adulto, cellule satelliti sono normalmente mitoticamente riposo 4-8. A seguito di infortunio, le cellule satellite sono attivati, avviare espressione di MyoD, e entrare nel ciclo cellulare per espandere la loro progenie, chiamato cellule precursori miogeniche o mioblasti 3. Dopo diversi cicli di divisione cellulare, mioblasti escono dal ciclo cellulare e fusibile tra loro al fine di differenziamento in miotubi multi-nucleate, seguite da fibre muscolari mature. Mioblasti isolati dal muscolo adulto possono essere facilmente espanse ex vivo. La capacità di mioblasti per diventare fibre muscolari in rigenerazione muscolare e perfibre muscolari ectopiche forma nei tessuti non muscolari è utilizzata da trapianto dei mioblasti, un potenziale approccio terapeutico per la distrofia muscolare di Duchenne (DMD) 4, disfunzioni urologiche 9, e insufficienza cardiaca 10. Infatti, mioblasti sono stati trapiantati con successo nel muscolo di entrambe mdx (modello DMD), topi e pazienti DMD 11-14. I mioblasti normali iniettati fondono con le fibre muscolari ospitante per migliorare l&#39;istologia e la funzione del muscolo malato. Il lavoro precedente ha dimostrato che sottopopolazioni di mioblasti sono più Stem Cell-like e rimangono in uno stato indifferenziato più a lungo nel muscolo durante la rigenerazione muscolare 5. Studi recenti hanno dimostrato che le cellule appena isolate satellitari di muscolo adulto contengono una popolazione di cellule simil-staminali che presenta attecchimento più efficiente e attività di auto-rinnovamento nella rigenerazione del muscolo 5-8. Pertanto, la purificazione di una popolazione pura di cellule satelliti quiescenti da adulto mu scheletricosclerosi è essenziale per la comprensione della biologia delle cellule satelliti, mioblasti e rigenerazione muscolare, e per lo sviluppo di terapie cellulari. Tuttavia, attuali metodi di purificazione potenziali di cellule satelliti quiescenti richiedono l&#39;uso di un costoso fluorescenza-attivato cell sorting (FACS) 1,2,6-8 macchina. Inoltre, l&#39;esposizione laser FACS tende ad indurre la morte cellulare durante la separazione, che causa bassa resa di cellule satelliti quiescenti 15. Qui, presentiamo un nuovo metodo per la purificazione rapido, economico e affidabile di cellule satelliti quiescenti di topo adulto muscolo scheletrico. Questo metodo utilizza dissociazione enzimatica seguita da cellule magnetico attivato classificare (MACS). Dopo aver isolato cellule satelliti quiescenti puri, queste cellule possono essere coltivate avere un gran numero di mioblasti dopo vari passaggi. Mostriamo anche che l&#39;iniezione intramuscolare di questi appena isolate cellule satellite quiescenti o ex vimioblasti vo espansi possono essere trapiantate in cardiotossina (CTX) indotta rigenerazione del mouse muscolo scheletrico per esaminare il contributo delle cellule derivate dal donatore alla rigenerazione delle fibre muscolari, nonché ai compartimenti cellulari satellitari per l&#39;esame delle attività di auto-rinnovamento.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Collagenase Type 2</td>
			<td>Worthington</td>
			<td>CLS-2</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Marigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen</td>
			<td>10569010</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>100 mg (3-4 mg/ml)</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>bFGF, human, Recombinant</td>
			<td>Gibco-Invitrogen</td>
			<td>PHG0263</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A5611-1G</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F10 Medium</td>
			<td>Gibco-Invitrogen</td>
			<td>11550-043</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>3600511</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco-Invitrogen</td>
			<td>26050088</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycin</td>
			<td>Gibco-Invitrogen</td>
			<td>15640055</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco-Invitrogen</td>
			<td>14190144</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco-Invitrogen</td>
			<td>25200072</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>18G needle with 12cc Syringe</td>
			<td>Fisher Scientific</td>
			<td>22-256-563</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#956;m)</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 ml tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10 ml disposable pipet</td>
			<td>BD Biosciences</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody-PE</td>
			<td>eBiosciences</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45 antibody-PE</td>
			<td>eBiosciences</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sca1 antibody-PE</td>
			<td>eBiosciences</td>
			<td>Dec-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;7 antibody</td>
			<td>MBL International</td>
			<td>ABIN487462</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini &#38; MidiMACS Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>LD Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma Aldrich</td>
			<td>C9759-1MG</td>
			<td>Stock 10 &#956;M in PBS</td>
		</tr>
		<tr>
			<td>31G Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>328438</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge (Microfuge 22R)</td>
			<td>Beckman Coulter</td>
			<td>368826</td>
			<td></td>
		</tr>
		<tr>
			<td>S241.5 Swinging Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>368882</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (Allegra X-22R)</td>
			<td>Beckman Coulter</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr>
			<td>Nod/Scid immunodeficient mice</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code 394</td>
			<td>Use 2 months old mice</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Name of the reagent</td>
			<td></td>
			<td></td>
			<td>Recipie</td>
		</tr>
		<tr>
			<td>10% and 2% FBS DMEM</td>
			<td></td>
			<td></td>
			<td>DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>0.2% Collagenase solution</td>
			<td></td>
			<td></td>
			<td>Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM.</td>
		</tr>
		<tr>
			<td>10% Matrigel solution</td>
			<td></td>
			<td></td>
			<td>Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20&#176;C until use.</td>
		</tr>
		<tr>
			<td>Matrigel-coated plate</td>
			<td></td>
			<td></td>
			<td>Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37&#176;C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20&#176;C for reusing.</td>
		</tr>
		<tr>
			<td>0.01% Collagen solution</td>
			<td></td>
			<td></td>
			<td>Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O.</td>
		</tr>
		<tr>
			<td>Collagen-coated plate</td>
			<td></td>
			<td></td>
			<td>Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months.</td>
		</tr>
		<tr>
			<td>bFGF stock solution</td>
			<td></td>
			<td></td>
			<td>bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 &#956;l in 500 &#956;l microcentrifuge tubes and kept in -80&#176;C.</td>
		</tr>
		<tr>
			<td>Myoblast medium</td>
			<td></td>
			<td></td>
			<td>500 mL HAM&#8217;S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 &#956;g of bFGF (20 &#956;l of bFGF stock).</td>
		</tr>
		<tr>
			<td>Differentiation medium</td>
			<td></td>
			<td></td>
			<td>500 mL DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>10 &#956;M Cardiotoxin stock</td>
			<td></td>
			<td></td>
			<td>1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS.</td>
		</tr>
	</tbody>
</table>

isolation, culture, and transplantation of muscle satellite cells

Cellule satelliti del muscolo sono una popolazione di cellule staminali necessarie per lo sviluppo post-natale del muscolo scheletrico e la rigenerazione, pari al 2-5% dei nuclei sublaminal nelle fibre muscolari. Nel muscolo adulto, cellule satelliti sono normalmente mitoticamente riposo. Dopo la lesione, tuttavia, le cellule satelliti iniziano proliferazione cellulare per produrre mioblasti, loro progenie, per mediare la rigenerazione del muscolo. Il trapianto di mioblasti derivati ​​dalle cellule satellite è stato ampiamente studiato come una possibile terapia per varie malattie, tra cui rigenerativi distrofia muscolare, insufficienza cardiaca e disfunzione urologica. Trapianto di mioblasti in distrofico muscoli scheletrici, cuore infartuato, e le disfunzioni vie urinarie ha dimostrato che i mioblasti engrafted possono differenziarsi in fibre muscolari nei tessuti dell&#39;ospite e visualizzare miglioramento funzionale parziale in queste malattie. Pertanto, lo sviluppo di metodi di purificazione efficiente di cellule satelliti quiescenti da muscl scheletricoe, così come la creazione di cellule derivate da colture di mioblasti satellitari e metodi di trapianto di mioblasti, sono essenziali per la comprensione dei meccanismi molecolari alla base di cellule auto-rinnovamento satellitare, attivazione e differenziazione. Inoltre, lo sviluppo di terapie cellulari per la distrofia muscolare e altre malattie rigenerative dipendono anche da questi fattori. Tuttavia, attuali metodi di purificazione potenziali di cellule satelliti quiescenti richiedono l&#39;uso di costosi fluorescenza-attivato cell sorting (FACS) macchine. Qui, presentiamo un nuovo metodo per la purificazione rapido, economico e affidabile di cellule satelliti quiescenti di topo adulto muscolo scheletrico per dissociazione enzimatica seguita da cellule magnetico attivato classificare (MACS). Dopo aver isolato cellule satelliti quiescenti puri, queste cellule possono essere coltivate avere un gran numero di mioblasti dopo vari passaggi. Questi appena isolatocellule satelliti quiescenti o ex vivo mioblasti espansi possono essere trapiantate in cardiotossina (CTX) indotta rigenerante topo muscoli scheletrici per esaminare il contributo delle cellule derivate dal donatore alla rigenerazione delle fibre muscolari, nonché compartimenti cellulari satellite per l&#39;esame di auto-rinnovamento attività.

Cellule satelliti quiescenti appena isolate mostrano un piccolo, forma rotonda (Figura 1G), e Pax7 espresso come marcatore definitivo per le cellule satellite quiescenti. Più del 90% delle cellule appena isolate esprimono Pax7 (Figura 1H e 1I). Maggior parte delle cellule contaminate sono da cellule del sangue che non crescono in modo efficiente in vitro seguente condizioni di coltura mioblasti. Così, mioblasti derivati ​​dalle cellule satelliti dominano ...

Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Isolation, Culture, and Transplantation of Muscle Satellite Cells

L&#39;isolamento e la coltura di una popolazione pura di cellule satelliti quiescenti, una popolazione di cellule staminali muscolari, è essenziale per la comprensione del muscolo biologia delle cellule staminali e rigenerazione, nonché trapianto di cellule staminali per terapie in distrofia muscolare e altre malattie degenerative.

Isolamento, Cultura e trapianto di cellule muscolari satellite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

isolation-culture-and-transplantation-of-muscle-satellite-cells

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Unit 1

Fibroblast Transformation and Muscle Stem Cells

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Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e trapianto di cellule staminali emopoietiche (CSE)

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Biologia

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Isolamento di cellule umane ombelicale muscolatura liscia arteriosa (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolamento e Cultura del mouse principale acinose pancreatiche Cellule

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolamento di polmonare cellule muscolari lisce di topi neonati

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e colture primarie di cellule di topo aortica endoteliali

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...