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
Description: 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

この研究は次の補助金によって財政上支えられる: 中国の自然科学基礎 (号 81572198;81772394 号)深セン大学 (第 2016031638); の高レベルの医療分野建設のための基金広東省、中国 (号の医学研究の基礎A2016314);深センの科学と技術のプロジェクト (No.JCYJ20170306092215436;違います。JCYJ20170412150609690;違います。JCYJ20170413161800287;違います。SGLH20161209105517753;違います。JCYJ20160301111338144)。

地域倫理委員会ガイドラインに従ってすべての動物実験を行い、動物のすべてのプロシージャは、機関動物ケアおよび使用委員会の深セン第二人民病院、深セン大学によって承認されました。
1. 分離し、rbSF MSCs の文化
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動物のプロシージャのための準備
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<li>RbSF のコレクションの骨格成熟女性ニュージーランド白ウサギを準備-MSCs。 1 日麻酔と穿?...

<ol>
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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. C. W., Prockop, D. J., Bunnell, B. A., Phinney, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+isolate+and+purify+human+bone+marrow+stromal+stem+cells.">A method to isolate and purify human bone marrow stromal stem cells.</a> Mesenchymal Stem Cells. , 45-57 (2008).</li><li>Krawetz, R. 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. 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=Modulating+chondrocyte+hypertrophy+in+growth+plate+and+osteoarthritic+cartilage.">Modulating chondrocyte hypertrophy in growth plate and osteoarthritic cartilage.</a> Journal of Musculoskeletal &amp; Neuronal Interact. 8 (4), 308 (2008).</li><li>Cooke, M. E., 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=Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+chondrocytes+promotes+chondrogenic+differentiation+without+hypertrophy.">Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy.</a> Osteoarthritis and Cartilage. 19 (10), 1210-1218 (2011).</li><li>Fischer, J., Dickhut, A., Rickert, M., Richter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+articular+chondrocytes+secrete+parathyroid+hormone-related+protein+and+inhibit+hypertrophy+of+mesenchymal+stem+cells+in+coculture+during+chondrogenesis.">Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.</a> Arthritis and Rheumatism. 62 (9), 2696-2706 (2010).</li></ol>

滑液の MSCs の存在は、細胞ベースの治療のための代替を提供します。以前の研究では、受傷後の期間5と相関する可能性があります彼らの滑液で間葉系幹細胞の多量を含む傷害のサイトを示しています。滑液の MSCs は、負傷18,19後自然治癒を高めるための組織に有益な可能性があります。SF MSCs の臨床応用は主な理由は関節で SF MSCs ...

MSCs は、特に軟骨病変のための再生医療のための貴重な情報源として提案されています。MSCs では、軟骨細胞、骨芽細胞、脂肪細胞、骨格筋細胞と内臓の間質細胞を含む広くその高膨張率と多系統分化潜在的な1のための幹細胞移植の領域を展開します。MSCs は、骨格筋、滑膜、骨髄、脂肪2,3,4から分離できます。調査結果にも、あるビジョ滑液の MSCs の存在と前の研究が関節再生5,6の有望な候補として滑液由来 Msc (SF MSCs) を発見します。
しかし、研究と人間のサンプルの臨床実験は多くの倫理的な問題の対象となります。代わりに、ウサギはされているし、MSCs の移植が軟骨の損傷を修復することができますを示すために最も一般的に使用される動物種であり続けます。近年、研究者数の増加は、両方生体外でウサギ間葉系幹細胞 (rbMSCs) を研究しているし生体内で、これらの細胞は、細胞生物学、組織生理学におけると同様に人間の MSCs。同様に、rbMSCs は人間の MSCs のように軸線維芽細胞の形態を表示するプラスチックの表面に付着することがあります。さらに、ウサギ間葉系サンプル、シンプルで簡単に7を入手します。さらに、最も重要なポイントは、CD44、CD90, CD105 などの表面マーカーを rbMSCs に表現として MSC 集団の同定の基準に一致してである多系統分化の能力が維持されること細胞療法8,9国際社会によって定義されます。特に、滑膜液 chondroprogenitors、非肥大軟骨分化表現型関節軟骨再生10、11、適切な細胞源のため TGF-β 1 による場合の対応 12。
しかし、SF MSCs の分離は臍帯、脂肪組織、末梢血、骨髄など、他の組織から大きく異なる。現在、浄化および SF MSCs の並べ替えのための最も一般的なアプローチはフローサイトメトリー ・免疫ビーズに基づく並べ替え、フローサイトメトリー法には、特定の環境と非常に高価な楽器13が必要ですが。
この記事は、白ウサギ ニュージーランドから滑液のサンプル集のシンプルで低侵襲の手順を示します。中には、rbSF MSCs 安定拡大培養CD90 正磁気ビーズによるプロシージャで分離されたし。最後に、プロトコルは、高純度と収穫の細胞源から生存率 MSCs を取得する方法を示します。
このプロトコルでは、形態、特定のマーカーと幹細胞の多能性の式に基づいて分離 rbSF MSCs が特徴です。CD45 と CD34 の式が負の値に対し、流れの cytometry ベース診療は CD44 と CD105 の重要な肯定的な表現を明らかにします。最後に、rbSF mscsの in vitroアッセイは、これらの細胞の骨、軟骨、脂肪細胞の分化を示しています。

<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

間葉系幹細胞 (Msc) は、細胞ベースの治療の主要な細胞ソースです。MSCs 関節腔滑液から軟骨組織工学用をされる可能性があります可能性があります。滑液 (SF MSCs) から MSCs は関節の再生のための有望な候補と考えられてきたし、彼らの潜在的な治療効果は重要な研究課題の後半。ニュージーランド白ウサギの膝関節腔から SF MSCs は人間の再生医療を評価するために最適化された並進モデルとして用いることができます。CD90 ベース磁気活性化細胞 (MAC) 技術を並べ替え、このプロトコルこの家兎モデルからウサギが SF MSCs (rbSF-MSCs) 正常取得ならびにさらに完全に区別するためにそれらを誘導することによってこれらの細胞の MSC の表現型を示します骨芽細胞、脂肪細胞、軟骨細胞。したがって、このアプローチは、細胞生物学の研究と簡単な装置や手順を使用してティッシュ エンジニア リングで適用できます。

分離・精製・ rbSF MSCs の文化: このプロトコルは、rbSF-MSCs MSC 表面マーカー CD90 の式に基づいて分離するのに MAC を使用します。RbSF MSCs の分離・精製とキャラクタリゼーション ・体外培養のプロトコルのプロセス フロー図を図 1に示します。
磁気活性化細胞と CD90 (MAC) を並...

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

磁気活性化細胞を分離し、滑膜液由来間葉系幹細胞が家兎モデルからの浄化戦略を並べ替え

この記事では、簡単な分離そしてニュージーランド白ウサギ滑液からの間葉系幹細胞の浄化のためのシンプルで経済的プロトコルを示します。

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

Research

JoVE Journal

Biology

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

ヘキストの分離および特性評価低 CD45負マウス肺間葉系幹細胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

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

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

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

Induction and Analysis of Epithelial to Mesenchymal Transition

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

スクレラクチニアン細胞集団の分離のための蛍光活性化細胞選別

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

3次元バイオリアクター培養による滑液間葉系幹細胞由来エクソソームの大規模調製

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

細胞ソーティングなしでスナップ凍結された不均一な組織サンプルから細胞種特異的RNAを単離(英語)

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...