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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Denne studien ble økonomisk støttet av følgende tilskudd: Natural Science Foundation i Kina (nr. 81572198; Nei 81772394); Fondet for høyt nivå medisinsk disiplin byggingen av Shenzhen University (nr. 2016031638); Medisinsk forskning grunnvoll Guangdong provinsen, Kina (nr. A2016314); og Shenzhen vitenskap og teknologi-prosjekter (nr. JCYJ20170306092215436; nei. JCYJ20170412150609690; nei. JCYJ20170413161800287; nei. SGLH20161209105517753; nei. JCYJ20160301111338144).

Alle dyreforsøk ble utført i henhold til regionale etikk veiledning, og alle dyr prosedyrer ble godkjent av institusjonelle Animal Care og bruk komiteen av Shenzhen andre folks Hospital, Shenzhen University.
1. Isoler og kultur av rbSF-MSCs
<ol><li>
Forberedelser for dyr prosedyren
	<ol>
<li>Forberede skjelettaktig eldre kvinnelige New Zealand white kaniner for innsamling av rbSF-MSCs. Utfør en klinisk undersøkelse av kaniner dagen før prosedyren anestesi og arthrocente...

<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>

Eksistensen av MSCs i synovial væsken gir et alternativ for cellen-basert terapi. Tidligere studier har vist at skade områder inneholder større mengder mesenchymal stamceller i deres synovial væske, som kan være positivt korrelert med etter skade perioden5. MSCs i synovial væske kan være gunstig for vev for å øke spontan helbredelse etter en skade18,19. Klinisk anvendelse av SF-MSCs har sjelden blitt dekket i litteraturen, hovedsa...

MSCs har blitt foreslått som en verdifull kilde for regenerativ medisin, spesielt for brusk lesjoner. MSCs, inkludert chondrocytes, osteoblasts, adipocytter, skjelettlidelser myocytter og visceral stromal celler, bredt utvide områdene for stilk cellen transplantasjon på grunn av deres høye ekspansjon rate og flere avstamning differensiering potensielle1. MSCs kan isoleres fra skjelettlidelser muskler, fagområder, benmargen og fettvev2,3,4. Funnene har også conﬁrmed tilstedeværelsen av MSCs i synovial væske, og tidligere forskning har identifisert synovial fluid-avledet MSCs (SF-MSCs) som lovende kandidater i articular gjenfødelse5,6.
Men er forskning og prekliniske eksperimentering på prøver fra mennesker underlagt mange etiske spørsmål. I stedet kanin har vært og fortsetter å være mest brukte dyrearter for å demonstrere at transplantasjon av MSCs kan reparere skader brusk. De siste årene, et økende antall forskere har studert kanin mesenchymal stamceller (rbMSCs) både i vitro og i vivo, disse cellene er ligner menneskelige MSCs i mobilnettet biologi og vev fysiologi. Tilsvarende er er rbMSCs i stand til å overholde plast overflater, vise spindel-fibroblast morfologi som menneskelige MSCs. Videre er kanin mesenchymal prøvene enkel og lett å få7. I tillegg er de viktigste punktene at rbMSCs express overflate markører, som CD44, CD90 og CD105, og at flere avstamning differensiering potensielle er bevart, som er i samsvar med kriteriene for identifikasjon av MSC populasjoner som definert av internasjonale samfunnet for mobilnettet terapi8,9. Spesielt er synovial væske chondroprogenitors i stand til ikke-hypertrofisk chondrogenesis når indusert av TGF-β1, dermed gjør dem egnet cellen kilder for svært articular brusk gjenfødelse10,11, 12.
Isolasjon av SF-MSCs er imidlertid svært forskjellig fra andre vev, inkludert navlestreng, fettvev, perifert blod og benmarg. Tiden, er de vanligste metodene for rensing og sortering av SF-MSCs flowcytometri og immunomagnetic perle-basert sortering, selv om flyt cytometri metoden krever et bestemt miljø og svært dyr instrumenter13.
Denne artikkelen presenterer en prosedyre for enkel og minimal invasiv innsamling av prøver av synovial væske fra New Zealand hvit kanin. Under inngrepet, rbSF-MSCs stabilt utvidet i vitro og deretter isolert CD90 positive magnetiske perle-baserte prosedyrer. Til slutt, protokollen viser hvordan å få MSCs med høy renhetsgrad og levedyktighet fra høstet celle kilder.
I denne protokollen kjennetegnes av isolerte rbSF-MSCs basert på deres morfologi, uttrykk for bestemte dataindikatorer og pluripotency for stamceller. Flow cytometri-baserte immunophenotyping avslører en betydelig positiv uttrykk for CD44 og CD105, mens uttrykk for CD45 og CD34 er negativt. Til slutt, i vitro analysen for rbSF-MSCs viser osteogenic, adipogenic og chondrogenic differensiering av disse cellene.

<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

Stamceller (MSCs) er den viktigste celle for celle-basert terapi. MSCs fra articular hulrom synovial væske kan potensielt brukes brusk vev engineering. MSCs fra synovial væske (SF-MSCs) har vært betraktet som lovende kandidater for articular gjenfødelse, og deres potensielle terapeutiske fordel har gjort dem en viktig forskning emne for sent. SF-MSCs fra i kneet hulrommet i New Zealand hvite kaninen kan anvendes som en optimalisert translasjonsforskning modell å vurdere menneskelige regenerativ medisin. Ved hjelp av CD90-baserte magnetiske aktivert celle sortering (Mac) teknologier, denne protokollen henter vellykket kanin SF-MSCs (rbSF-MSCs) fra denne kanin modellen og ytterligere viser fullt MSC fenotypen av disse cellene av til å differensiere å osteoblasts, adipocytter og chondrocytes. Derfor kan denne tilnærmingen brukes i biologi forskning og vev engineering bruker enkel utstyr og prosedyrer.

Isolasjon, rensing og kultur av rbSF-MSCs: Denne protokollen bruker Mac for å isolere rbSF-MSCs, basert på uttrykket av MSC overflaten merket CD90. Et prosessflytdiagram rbSF-MSCs' isolasjon, rensing, og karakterisering og i vitro kultur protokollen er vist i figur 1.
Celle morfologi etter magnetiske aktivert celle sortering (Mac) med CD90: For det ...

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

Magnetisk-aktivert celle sortering strategier for å isolere og rense Synovial Fluid-avledet Mesenchymal stamceller fra en kanin modell

Denne artikkelen presenterer en enkel og økonomisk protokoll for enkel isolasjon og rensing av mesenchymal stamceller fra New Zealand hvit kanin synovial væske.

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

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

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

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

Isolasjon &amp; Karakterisering av Hoechst Lav CD45 Negative Mus Lung stamceller

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

Isolasjon og kultur av individuelle myofibers og deres satellitt celler fra voksne 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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Induksjon og analyse av Epiteliale til Mesenchymale Transition

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

Nyreskade Mikroinjeksjon i Sebrafisk til Model Akutt nyreskade

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

Fluorescensaktivert cellesortering for isolering av sklerractiniske cellepopulasjoner

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

Storskala fremstilling av synovialvæske mesenkymale stamcelleavledede eksosomer ved 3D bioreaktorkultur

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

Isoler celletypespesifikke RNAer fra snapfrosne heterogene vevsprøver uten cellesortering

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