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

This study was financially supported by the following grants: the Natural Science Foundation of China (No. 81572198; No. 81772394); the Fund for High Level Medical Discipline Construction of Shenzhen University (No. 2016031638); the Medical Research Foundation of Guangdong Province, China (No. A2016314); and Shenzhen Science and Technology Projects (No. JCYJ20170306092215436; No. JCYJ20170412150609690; No. JCYJ20170413161800287; No. SGLH20161209105517753; No. JCYJ20160301111338144).

All animal experiments were conducted in accordance with the regional Ethics Committee guidelines, and all animal procedures were approved by the Institutional Animal Care and Use Committee of Shenzhen Second People&#39;s Hospital, Shenzhen University.
1. Isolate and Culture the rbSF-MSCs
<ol>
	<li>Preparations for the animal procedure
	<ol>
		<li>Prepare skeletally-mature female New Zealand white rabbits for the collection of rbSF-MSCs. Perform a clinical examination of the...

<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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+mesenchymal+stem+cells+from+adult+human+synovial+membrane.">Multipotent mesenchymal stem cells from adult human synovial membrane.</a> Arthritis Rheumatology. 44 (8), 1928-1942 (2001).</li><li>Zuk, P. 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>

The existence of MSCs in synovial fluid provides an alternative for cell-based therapy. Previous studies have shown that injury sites contain higher amounts of mesenchymal stem cells in their synovial fluid, which may be positively correlated with the post-injury period5. The MSCs in synovial fluid may be beneficial to tissue for enhancing the spontaneous healing after an injury18,19. The clinical application of SF-MSCs has rarely been cov...

MSCs have been suggested as a valuable source for regenerative medicine, especially for cartilage lesions. MSCs, including chondrocytes, osteoblasts, adipocytes, skeletal myocytes, and visceral stromal cells, broadly expand the areas for stem cell transplantation due to their high expansion rate and multi-lineage differentiation potential1. MSCs can be isolated from the skeletal muscle, synovium, bone marrow, and adipose tissue2,3,4. Findings have also con&#64257;rmed the presence of MSCs in synovial fluid, and previous research has identified synovial fluid-derived MSCs (SF-MSCs) as promising candidates for articular regeneration5,6.
However, research and preclinical experimentation on human samples are subject to many ethical issues. Instead, rabbits have been and continue to be the most commonly used animal species to demonstrate that transplantation of MSCs can repair cartilage damage. In recent years, an increasing number of researchers have studied rabbit mesenchymal stem cells (rbMSCs) both in vitro and in vivo, as these cells are similar to human MSCs in their cellular biology and tissue physiology. Similarly, the rbMSCs are capable of adhering to plastic surfaces, displaying spindle-fibroblast morphology as in human MSCs. Furthermore, rabbit mesenchymal samples are simple and easy to obtain7. Additionally, the most crucial points are that rbMSCs express surface markers, such as CD44, CD90, and CD105, and that the multi-lineage differentiation potential is preserved, which is in agreement with the criteria for identification of MSC populations as defined by the International Society for Cellular Therapy8,9. In particular, synovial fluid chondroprogenitors are capable of non-hypertrophic chondrogenesis when induced by TGF-&#946;1, thus making them suitable cell sources for phenotypically articular cartilage regeneration10,11,12.
However, the isolation of SF-MSCs is greatly different from other tissues, including the umbilical cord, adipose tissue, peripheral blood, and bone marrow. Currently, the most common approaches for the purification and sorting of SF-MSCs are flow cytometry and immunomagnetic bead-based sorting, although the flow cytometry method requires a specific environment and highly expensive instruments13.
This article presents a procedure for the simple and minimally invasive collection of samples of synovial fluid from New Zealand white rabbits. During the procedure, the rbSF-MSCs are stably expanded in vitro and then isolated with CD90 positive magnetic bead-based procedures. Finally, the protocol shows how to obtain MSCs with a high purity and viability from the harvested cell sources.
In this protocol, the isolated rbSF-MSCs are characterized based on their morphology, expression of specific markers, and pluripotency for stem cells. Flow cytometry-based immunophenotyping reveals a significant positive expression of CD44 and CD105, whereas the expression of CD45 and CD34 is negative. Finally, an in vitro assay for rbSF-MSCs demonstrates the osteogenic, adipogenic, and chondrogenic differentiation of these cells.

<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

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

Isolation, Purification, and Culture of the rbSF-MSCs: 
This protocol uses MACS to isolate rbSF-MSCs, based on the expression of the MSC surface marker CD90. A process flow diagram of rbSF-MSCs&#39; isolation, purification, and characterization and the in vitro culture protocol is shown in Figure 1.
Cell Morphology after Magnetic Activated Cell Sorting (MACS) w...

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

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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

This method can help answer key questions in the sorting protocol in the stem cell field by providing a procedure for purifying mesenchymal stem cells directly from synovial fluid. The main advantage of this technique is that we have establish a simple and economic protocol for the isolation and the purification of MACS from rabbit synovial fluid. Generally in the research, due to this method, we make great efforts, because plurating of cell population is dependent on producing a single cell suspension.We first had the idea for this method when we noticed that only the magnetic-activated cell sorting method is currently approved by the FDA for clinical applications. Begin this procedure with animal anesthesia as described in the text protocol. To perform collection of articular synovial fluid from the knee of the rabbit, select an area about five by five centimeters in size around the knee.Shave the rabbit hair from around this area using a safety electric shaver. Alternately, disinfect the procedure site with povidone iodine solution and 75%ethanol three times. Then, apply sterile drapes after area has thoroughly dried.Use a sterile hypodermic syringe to inject one to two milliliters of isotonic saline solution into the knee joint cavity from the lateral articular space. Move the knee three to four times, and then aspirate all the synovial fluid at room temperature. Filter the synovial fluid through a 40 micron nylon cell strainer to remove any debris, within four hours.To culture the rabbit SF-MSCs, collect the filtered fluid in 50 milliliter centrifuge tubes, and centrifuge at 1, 500 rpm for ten minutes at room temperature. Discard the supernatant after the centrifugation and wash the pellet with PBS. After resuspending the pellet with the complete culture medium, plate the medium in 100 milliliter dishes.Incubate the dishes at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide. After 48 hours, replenish the dishes with fresh medium to remove any non-adherent cells. Replace the medium twice a week for two weeks as passage zero.After 14 days following the initial plating, many colonies should've formed in the culture dishes. Select the colonies larger than two millimeters in diameter. Mark where the selected colonies are located, by tracing their circumference on the dish bottom.Discard the colonies less than two millimeters wide using cell scrapers. Digest the selected colonies with about five microliters of 0.25%trypsin using a cloning cylinder, and transfer the colonies to a new dish to passage. When the cells reach about 80%confluency, aspirate the medium.Then, add one to two milliliters of 0.25%trypsin EDTA to each dish. Incubate the dishes for two to three minutes to allow cell detachment. Once the cells are detached, add an equal amount of the culture medium to inactive the trypsin.Pass the cell suspension through a 40 micron cell strainer and collect the filtrate in a 15 milliliter tube. Then spin down the cells at 600 times G for ten minutes at room temperature. Resuspend the cell pellet in MACS Running Buffer.To perform magnetic labeling, determine the cell number using a hemocytometer. Centrifuge the cell suspension at 300 times G for ten minutes at four degrees Celsius. After completely aspirating the supernatant, add 80 microliters of resuspension buffer per ten million total cells.For ten million total cells, add 20 microliters of microbeads conjugated with a monoclonal anti-rabbit CD90 antibody. Mix the magnetic beads and cells evenly in the tubes. Then incubate them at four degrees Celsius for 15 minutes in the dark.Add one milliliter of buffer per ten million cells to the tube, and then centrifuge at 300 times G for ten minutes at four degrees Celsius, to wash the cells. Discard the supernatant after centrifugation. Resuspend the pellet in 500 microliters of buffer per ten million cells.Place the column, with the column wings to the front, into the magnetic field of the magnetic separator. Rinse the magnetic separator column with 500 microliters of the buffer per ten million cells. Transfer the single cell suspension into the column.Let the negative cells pass through the magnetic field to discard these unlabeled cells. Wash the column one to two times with 500 microliter of the buffer per ten million cells, and discard the flow through. Transfer the column into a 15 milliliter centrifuge tube.Add one milliliter of the buffer per ten million cells to the column. Then immediately push the plunger into the column, to flush out the magnetically labeled cells. Repeat the aforementioned procedure with a second magnetic separator column, to increase the purity of CD90-positive cells, which can enrich the eluted fraction.After centrifuging the cell suspension at 300 times G for ten minutes, aspirate the supernatant, and resuspend the pellet with the culture medium. Inoculate the cells in 100 milliliter dishes after the magnetic-activated cell sorting. Incubate the dishes at 37 Celsius in a humidified cell incubator with 5%carbon dioxide.When 80 to 90%confluence of the primary culture is achieved, at about seven to ten days, digest the adherent cells with 0.25%trypsin EDTA and passage them at a one to two dilution to make passage two. Use the same method to pass the cells to passage three, which can be used for the in-vitro assays. Shown here, is the morphology of the monolayer cultured rabbit SF-MSCs before and after MACS, under an inverted microscope.Before sorting, the adherent rabbit synovial fluid cell populations display heterogeneity. They contain diverse cell types and sizes such as oval, long-spindle, short-spindle, and stellate morphology. The main morphology of passage two and passage six, is a spindle morphology.Following MACS with CD90, the cell populations exhibit a homogenous morphology. With the sub culture, the cell number increases. Flow cytometry analysis demonstrated that rabbit SF-MSC cells were positive for the MSC markers CD44 and CD105.While negative for the endothelial cell marker CD34, and for the hematopoietic cell marker CD45. The results showed that before MACS, the rabbit SF-MSC population was composed of approximately 40%MSC cells. After MACS, the enriched population contained greater than 99%MSC cells.Shown here, is flow cytometry analysis of the CD90-positive marker before and after MAC sorting. Alizarin Red staining demonstrated that mineralized nodules formed under osteogenic induction for three weeks. After three weeks of adipogenic induction, accumulation of lipid rich vacuoles was detected by intracellular Oil Red O staining.After tree weeks of chondrogenic induction, the cell pellet was histologically assayed with Toluidine Blue staining. The positive acidic proteoglycan characterized the chondrocyte-like cells. After three weeks of induction, the relative mRNA expression of the osteoblast marker, the adipogenic marker and the chondrogenic marker was detected.Once mastered, this technique can be done in two to four hours. After watching this video, you should have a good understanding of the straight forward isolation and the purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid. While attempting this procedure it's important to remember to select those colonies larger than two milliliters in diameter.Also produce a single cell suspension before immunomagnetic sorting, as unwanted cells attached to a labeled cell will remain on the magnetic bead. Following this procedure, other methods like fluorescence activated cell sorting can be performed in order to answer additional questions addressing the safety and efficacy of purification of mesenchymal stem cells. After it's development, this technique paved the way for researchers in the field of articular cartilage regeneration to explore the ideal CD cells in articular cartilage repair.Don't forget that the operations were performed on arch cooling bench, and the precautions such as gloves, should be always be taken while performing this procedure. The implications of this technique extend towards therapy of articular cartilage injury, because synovial fluid derived MASC are promising candidates for articular cartilage regeneration.

Research

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