Name: magnetic-activated cell sorting strategies to isolate and purify synovial fluid-derived mesenchymal stem cells from a rabbit model
Uploaded: 2018-08-10T14:00:00
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Cette étude a été financée par les subventions suivantes : la Fondation des sciences naturelles de Chine (no 81572198 ; No. 81772394) ; le Fonds pour la Construction de haute niveau Discipline médicale de l’Université de Shenzhen (n° 2016031638) ; la Fondation de recherche médicale de la Province de Guangdong, Chine (no. A2016314) ; et Shenzhen projets Science et technologie (no. JCYJ20170306092215436 ; Lol JCYJ20170412150609690 ; Lol JCYJ20170413161800287 ; Lol SGLH20161209105517753 ; Lol JCYJ20160301111338144).

Toutes les expériences sur des animaux ont été menées selon les directives d’éthique Comité régionales, et toutes les procédures d’animaux ont été approuvées par l’animalier institutionnel et utilisation Comité Shenzhen deuxième populaire de l’hôpital, Université de Shenzhen.
1. isoler et Culture les rbSF-CSM
<ol><li>
Préparatifs de la procédure de l’animale
	<ol>
<li>Préparer le squelette-mature femme Nouvelle Zélande lapins blancs pour la colle...

<ol>
	<li>Oreffo, R. O., Cooper, C., Mason, C., Clements, M. <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:+lineage,+plasticity,+and+skeletal+therapeutic+potential.">Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential.</a> Stem Cell Reviews. 1 (2), 169-178 (2005).</li><li>Asakura, A., Rudnicki, M. A., Komaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells+are+multipotential+stem+cells+that+exhibit+myogenic,+osteogenic,+and+adipogenic+differentiation.">Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation.</a> Differentiation. 68 (4-5), 245-253 (2001).</li><li>De, B. C., Dell'Accio, F., Tylzanowski, P., Luyten, F. 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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=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Molecular Biology of the Cell. 13 (12), 4279-4295 (2002).</li><li>McGonagle, D., Jones, E., English, A., Emery, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+and+phenotypic+characterization+of+synovial+fluid+multipotential+mesenchymal+progenitor+cells+in+inflammatory+and+degenerative+arthritis.">Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis.</a> Arthritis &amp; Rheumatism. 50 (3), 817-827 (2004).</li><li>Jia, Z., 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+and+characterisation+of+human+mesenchymal+stem+cells+derived+from+synovial+fluid+by+magnetic+activated+cell+sorting+(MACS).">Isolation and characterisation of human mesenchymal stem cells derived from synovial fluid by magnetic activated cell sorting (MACS).</a> Cell Biology International. , (2017).</li><li>Bashir, 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,+culture+and+characterization+of+New+Zealand+white+rabbit+mesenchymal+stem+cells+derived+from+bone+marrow.">Isolation, culture and characterization of New Zealand white rabbit mesenchymal stem cells derived from bone marrow.</a> Asian Journal of Animal &amp; Veterinary Advances. 10 (8), 13-30 (2015).</li><li>Song, 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=Differentiation+potential+of+rabbit+CD90-positive+cells+sorted+from+adipose-derived+stem+cells+in+vitro.">Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 53 (1), 1-6 (2016).</li><li>Lee, T. 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=Comparison+of+surface+markers+between+human+and+rabbit+mesenchymal+stem+cells.">Comparison of surface markers between human and rabbit mesenchymal stem cells.</a> PLOS One. 9 (11), e111390 (2014).</li><li>Stewart, M. C., Chen, Y., Bianchessi, M., Pondenis, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+equine+synovial+fluid-derived+chondroprogenitor+cells.">Phenotypic characterization of equine synovial fluid-derived chondroprogenitor cells.</a> Stem Cell Biology and Research. 3 (1), (2016).</li><li>Prado, A. A. F., 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=Characterization+of+mesenchymal+stem+cells+derived+from+the+equine+synovial+fluid+and+membrane.">Characterization of mesenchymal stem cells derived from the equine synovial fluid and membrane.</a> BioMed Central Veterinary Research. 11 (1), 281 (2015).</li><li>Yoshimura, 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=Comparison+of+rat+mesenchymal+stem+cells+derived+from+bone+marrow,+synovium,+periosteum,+adipose+tissue,+and+muscle.">Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle.</a> Cell and Tissue Research. 327 (3), 449-462 (2007).</li><li>Wu, C. 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=Intra-articular+injection+of+platelet-rich+fibrin+releasates+in+combination+with+bone+marrow-derived+mesenchymal+stem+cells+in+the+treatment+of+articular+cartilage+defects:+an+in+vivo+study+in+rabbits.">Intra-articular injection of platelet-rich fibrin releasates in combination with bone marrow-derived mesenchymal stem cells in the treatment of articular cartilage defects: an in vivo study in rabbits.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 105 (6), 1536-1543 (2017).</li><li>John, T., Lerche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anesthesia and Analgesia for Veterinary Technicians. , (2011).</li><li>Koyama, N., 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=Pluripotency+of+mesenchymal+cells+derived+from+synovial+fluid+in+patients+with+temporomandibular+joint+disorder.">Pluripotency of mesenchymal cells derived from synovial fluid in patients with temporomandibular joint disorder.</a> Life Sciences. 89 (19-20), 741-747 (2011).</li><li>Kim, Y. 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=Isolation+and+characterization+of+human+mesenchymal+stem+cells+derived+from+synovial+fluid+in+patients+with+osteochondral+lesion+of+the+talus.">Isolation and characterization of human mesenchymal stem cells derived from synovial fluid in patients with osteochondral lesion of the talus.</a> American Journal of Sports Medicine. 43 (2), 399-406 (2015).</li><li>Vereb, Z., 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=Immunological+properties+of+synovial+fluid-derived+mesenchymal+stem+cell-like+cells+in+rheumatoid+arthritis.">Immunological properties of synovial fluid-derived mesenchymal stem cell-like cells in rheumatoid arthritis.</a> Annals of the Rheumatic Diseases. 74 (Suppl 1), A64-A65 (2015).</li><li>Matsukura, Y., Muneta, T., Tsuji, K., Koga, H., Sekija, I. <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+in+synovial+fluid+increase+after+meniscus+injury.">Mesenchymal stem cells in synovial fluid increase after meniscus injury.</a> Clinical Orthopaedics &amp; Related Research. 472 (5), 1357-1364 (2014).</li><li>Morito, T., 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=Synovial+fluid-derived+mesenchymal+stem+cells+increase+after+intra-articular+ligament+injury+in+humans.">Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans.</a> Rheumatology. 47 (8), 1137-1143 (2008).</li><li>Hegewald, A. 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=Hyaluronic+acid+and+autologous+synovial+fluid+induce+chondrogenic+differentiation+of+equine+mesenchymal+stem+cells:+a+preliminary+study.">Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study.</a> Tissue &amp; Cell. 36 (6), 431-438 (2004).</li><li>Jones, E. 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=Synovial+fluid+mesenchymal+stem+cells+in+health+and+early+osteoarthritis:+detection+and+functional+evaluation+at+the+single+cell+level.">Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single cell level.</a> Arthritis &amp; Rheumatism. 58 (6), 1731-1740 (2008).</li><li>Makker, K., Agarwal, A., Sharma, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+activated+cell+sorting+(MACS):+utility+in+assisted+reproduction.">Magnetic activated cell sorting (MACS): utility in assisted reproduction.</a> Indian Journal of Experimental Biology. 46 (7), 491-497 (2008).</li><li>Schmitz, B., 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=Magnetic+activated+cell+sorting+(MACS)+&#8212;+a+new+immunomagnetic+method+for+megakaryocytic+cell+isolation:+comparison+of+different+separation+techniques.">Magnetic activated cell sorting (MACS) &#8212; a new immunomagnetic method for megakaryocytic cell isolation: comparison of different separation techniques.</a> European Journal of Haematology. 52 (5), 267-275 (1994).</li><li>Reinhardt, M., Bader, A., Giri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Devices+for+stem+cell+isolation+and+delivery:+current+need+for+drug+discovery+and+cell+therapy.">Devices for stem cell isolation and delivery: current need for drug discovery and cell therapy.</a> Expert Review of Medical Devices. 12 (3), 353-364 (2015).</li><li>Gronthos, S., Zannettino, A. 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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=Synovial+fluid+progenitors+expressing+CD90++from+normal+but+not+osteoarthritic+joints+undergo+chondrogenic+differentiation+without+micro-mass+culture.">Synovial fluid progenitors expressing CD90+ from normal but not osteoarthritic joints undergo chondrogenic differentiation without micro-mass culture.</a> PLOS One. 7 (8), e43616 (2012).</li><li>Ogata, 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=Purified+human+synovium+mesenchymal+stem+cells+as+a+good+resource+for+cartilage+regeneration.">Purified human synovium mesenchymal stem cells as a good resource for cartilage regeneration.</a> PLOS One. 10 (6), e0129096 (2015).</li><li>Dominici, 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells:+the+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Gauci, S. 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L’existence de MSCs dans le liquide synovial offre une alternative pour la thérapie cellulaire. Des études antérieures ont montré que les blessures sites contiennent des quantités plus élevées de cellules souches mésenchymateuses dans leur liquide synovial, qui peuvent être positivement corrélée avec la période post-traumatique5. Les MSCs dans le liquide synovial peuvent être bénéfiques au tissu pour améliorer la cicatrisation spontanée après une blessure18</s...

MSCs ont été proposées comme une source précieuse pour la médecine régénérative, surtout pour les lésions du cartilage. MSCs, y compris les chondrocytes, ostéoblastes, adipocytes, myocytes squelettiques et viscérales cellules stromales, étendre largement les zones destinés à la transplantation de cellules souches en raison de leur forte expansion taux et lignées multiples différenciation possibles1. MSCs peuvent être isolés dans le squelette musculaire, membrane synoviale, la moelle osseuse et le tissu adipeux2,3,4. Résultats ont conﬁrmé aussi la présence de MSCs dans le liquide synovial, et des recherches antérieures a identifié MSCs de dérivés de liquide synoviales (SF-MSCs) comme candidats prometteurs pour régénération articulaire5,6.
Cependant, recherche et expérimentation préclinique sur échantillons humains sont soumis à nombreuses questions éthiques. Au lieu de cela, les lapins ont été et continuent d’être le plus couramment utilisé des espèces animales pour démontrer que la transplantation de MSCs peut réparer les lésions du cartilage. Ces dernières années, un nombre croissant de chercheurs ont étudié les cellules souches mésenchymateuses de lapin (rbMSCs) les deux in vitro et in vivo, que ces cellules sont semblables aux humains MSCs dans leur physiologie cellulaire de la biologie et de tissus. De même, les rbMSCs sont capables d’adhérer aux surfaces plastiques, affichant la morphologie de la broche-fibroblaste comme dans MSCs humaines. En outre, les échantillons mésenchymateuses lapin sont simples et faciles à obtenir7. En outre, les points cruciaux sont que rbMSCs expriment des marqueurs de surface, telles que CD44 CD90 et CD105, et que la différenciation des lignées multiples potentielle est préservée, ce qui est en accord avec les critères d’identification des populations de MSC comme défini par la société internationale de thérapie cellulaire8,9. En particulier, chondroprogenitors fluides synoviales sont capables de non-hypertrophique chondrogenèse lorsque induite par le TGF-β1, rendant les sources cellulaire appropriée pour le cartilage articulaire phénotypiquement régénération10,11, 12.
Toutefois, l’isolement de SF-MSCs est très différent des autres tissus, y compris le cordon ombilical, le tissu adipeux, sang périphérique et la moelle osseuse. Actuellement, les approches courantes pour la purification et le tri des SF-MSCs sont cytométrie en flux et immunomagnetic axée sur la perle tri, bien que la méthode de cytométrie de flux nécessite un environnement spécifique et très coûteux instruments13.
Cet article présente une procédure simple et peu invasive recueillir des échantillons de liquide synovial de Nouvelle Zélande lapins blancs. Au cours de la procédure, les rbSF-MSCs sont solidement développé in vitro et ensuite isolement avec CD90 positives procédures fondées sur les perles magnétiques. Enfin, le protocole montre comment obtenir les MSCs avec une grande pureté et de la viabilité dans les sources de cellules récoltées.
Dans le présent protocole, les rbSF-CSM isolées sont caractérisés basé sur leur morphologie, l’expression de marqueurs spécifiques et la pluripotence des cellules souches. Immunophénotypage axée sur la cytométrie de flux révèle une expression positive significative du CD44 et CD105, tandis que l’expression de CD45 et CD34 est négative. Enfin, un essai in vitro avec de rbSF-MSCs montre la différenciation ostéogénique, adipocytaire et chondrogéniques de ces cellules.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MesenGro</td>
			<td>StemRD</td>
			<td>MGro-500 1703</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>MesenGro Supplement</td>
			<td>StemRD</td>
			<td>MGro-500 M1512</td>
			<td>Component of MSCs culture medium</td>
		</tr>
		<tr>
			<td>DMEM basic</td>
			<td>Gibco Inc.</td>
			<td>C11995500BT</td>
			<td>MSCs differentiation medium</td>
		</tr>
		<tr>
			<td>Isotonic saline solution</td>
			<td>Litai, China</td>
			<td>5217080305</td>
			<td>Cavity arthrocentesis procedure reagent</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>HyClone Inc.</td>
			<td>SH30256.01B</td>
			<td>PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Inc.</td>
			<td>10099-141</td>
			<td>Component of MSCs culture medium</td>
		</tr>
		<tr>
			<td>Povidone iodine solution</td>
			<td>Guangdong, China</td>
			<td>150605</td>
			<td>Sterilization agent</td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Lircon, china</td>
			<td>170917</td>
			<td>Sterilization agent</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco Inc.</td>
			<td>25200-056</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr>
			<td>1% Penicillin-Streptomycin</td>
			<td>Gibco Inc.</td>
			<td>15140-122</td>
			<td>Component of MSCs medium</td>
		</tr>
		<tr>
			<td>MACS Running Buffer</td>
			<td>MiltenyiBiotec</td>
			<td>5160112089</td>
			<td>Containing phosphate-buffered saline (PBS), 0.5% bovine serum albumin(BSA), and 2 mMEDTA</td>
		</tr>
		<tr>
			<td>CD90 antibody conjugated MicroBeads</td>
			<td>MiltenyiBiotec</td>
			<td>5160801456</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td>Component of MSCs osteogenic differentiation</td>
		</tr>
		<tr>
			<td>ITS</td>
			<td>BD</td>
			<td>354352</td>
			<td>1%, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma-Aldrich</td>
			<td>P5607</td>
			<td>0.35 mM, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>L-ascorbic acid-2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>50 mM, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>0.5 mM, Component of adipogenic differentiation</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td>100 mM, Component of adipogenic differentiation</td>
		</tr>
		<tr>
			<td>TGF&#946;1</td>
			<td>Peprotech</td>
			<td>100-21</td>
			<td>10 ng/mL, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>&#945;-glycerophsphate</td>
			<td>Sigma-Aldrich</td>
			<td>G6751</td>
			<td>Component of MSCs osteogenic differentiation</td>
		</tr>
		<tr>
			<td>CD34 Polyclonal Antibody, FITC Conjugated</td>
			<td>Bioss</td>
			<td>bs-0646R-FITC</td>
			<td>Hematopoietic stem cells marker</td>
		</tr>
		<tr>
			<td>Mouse antirabbit CD44</td>
			<td>Bio-Rad</td>
			<td>MCA806GA</td>
			<td>Thy-1 membrane glycoprotein (MSCs marker)</td>
		</tr>
		<tr>
			<td>CD45 (Monoclonal Antibody)</td>
			<td>Bio-Rad</td>
			<td>MCA808GA</td>
			<td>Hematopoietic stem cells marker</td>
		</tr>
		<tr>
			<td>CD105 antibody</td>
			<td>Genetex</td>
			<td>GTX11415</td>
			<td>MSCs marker</td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9030</td>
			<td>Precipitates RNA extraction organic phases</td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Wenge, China</td>
			<td>61553</td>
			<td>Extract total RNA</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td>Isolate total RNA</td>
		</tr>
		<tr>
			<td>SYBR green master mix</td>
			<td>Takara Bio, Japan</td>
			<td>RR420A</td>
			<td>PCR test</td>
		</tr>
		<tr>
			<td>cDNA synthesis kit</td>
			<td>Takara Bio, Japan</td>
			<td>RR047A</td>
			<td>Reverse-transcribed to complementary DNA</td>
		</tr>
		<tr>
			<td>Alizarin Red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td>Staining of calcium compounds</td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Sigma-Aldrich</td>
			<td>89640</td>
			<td>Staining of cartilaginous tissue</td>
		</tr>
		<tr>
			<td>Oil Red O solution</td>
			<td>Sigma-Aldrich</td>
			<td>O1391L</td>
			<td>Lipid vacuole staining</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>MiltenyiBiotec</td>
			<td>130-042-102</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>MultiStand</td>
			<td>MiltenyiBiotec</td>
			<td>130-042-303</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>MiltenyiBiotec</td>
			<td>130-042-201</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>FALCON Inc.</td>
			<td>352340</td>
			<td>40 &#956;m nylon</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>ISOLAB Inc.</td>
			<td>075.03.001</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Falcon 100 mm&#160; dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td>Cell culture dish</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>RNA Extraction and PCR</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Sigma-Aldrich</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>Centrifuge cells</td>
		</tr>
		<tr>
			<td>Carbon dioxide cell incubator</td>
			<td>Thermo scientific</td>
			<td>3111</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Real-Time PCR Instrument</td>
			<td>Life Tech</td>
			<td>QuantStudio</td>
			<td>Real-Time quantitative polymerase chain reaction</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>342975</td>
			<td>Cell analyzer</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf</td>
			<td>O25456F</td>
			<td>Transfer the liquid</td>
		</tr>
		<tr>
			<td>Cloning cylinder</td>
			<td>Sigma-Aldrich</td>
			<td>C3983-50EA</td>
			<td>Isolate and pick individual cell colonies</td>
		</tr>
		<tr>
			<td>Sterile hypodermic syringe</td>
			<td>Double-Dove, China</td>
			<td>131010</td>
			<td>Arthrocentesis procedure</td>
		</tr>
		<tr>
			<td>Rabbit cage</td>
			<td>Zhike, China</td>
			<td>ZC-TGD</td>
			<td>Restrain the rabbit</td>
		</tr>
	</tbody>
</table>

magnetic-activated cell sorting strategies to isolate and purify synovial fluid-derived mesenchymal stem cells from a rabbit model

Cellules souches mésenchymateuses (CSM) sont la source principale de cellule pour la thérapie cellulaire. MSCs du fluide synovial de la cavité articulaire susceptibles de servir pour l’ingénierie tissulaire du cartilage. MSCs de liquide synovial (SF-MSCs) ont été considérés comme des candidats prometteurs pour la régénération articulaire, et leur bénéfice thérapeutique potentiel a fait d’eux un sujet de recherche importants de la fin. SF-MSCs de la cavité du genou les lapins blancs de Nouvelle-Zélande peuvent être utilisés comme un modèle optimisé translationnel afin d’évaluer la médecine régénérative humaine. Au moyen de base CD90 cell activé magnétique tri technologies (MACS), ce protocole avec succès obtient lapin SF-MSCs (rbSF-MSCs) de ce modèle de lapin et plus pleinement montre le phénotype de la maîtrise de ces cellules par incitant à faire la différence à ostéoblastes, les adipocytes et les chondrocytes. Par conséquent, cette approche peut être appliquée dans la recherche en biologie cellulaire et ingénierie tissulaire en utilisant des procédures et des équipements simples.

Isolement, Purification et la Culture des rbSF-MSCs : Ce protocole utilise des MACS d’isoler rbSF-MSCs, basés sur l’expression du marqueur de surface MSC CD90. Un diagramme de flux de processus d’isolement des rbSF-MSCs, purification et la caractérisation et le protocole de culture in vitro est illustré à la Figure 1.
Morphologie des cellules après de...

Watch this Scientific Journal Video about Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model at JoVE.com

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Cellule magnétique activé tri des stratégies visant à isoler et purifier Synovial fluide mésenchymateuses cellules souches dérivées d’un modèle de lapin

Cet article présente un protocole simple et économique pour la simple isolation et la purification de cellules souches mésenchymateuses du liquide synovial de Nouvelle-Zélande lapin blanc.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

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