Name: isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs
Uploaded: 2016-07-02T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

We gratefully acknowledge the financial support provided by the Next-Generation BioGreen 21 Program (No. PJ007969), Rural Development Administration, and the National Research Foundation (grant no. NRF-2015R1D1A1A01056639) of the Republic of Korea.

Experimentos em animais foram autorizados pelo Centro animal for Biomedical Experimentação da Universidade Nacional de Gyeongsang. 1. Preparativos para o Procedimento de animal <ol><li> Prepara-se o adulto porquinhos do sexo feminino para a recolha não-invasivo de MSCs a partir de medula óssea e no fluido sinovial. Realizar o exame clínico dos porquinhos um dia antes da anestesia e coleta de amostras. <ol><li> Fornecer animais clinicamente normais de exames físicos, que incluem a tem...

<ol>
	<li>Woodbury, D., Schwarz, E. J., Prockop, D. J., Black, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+and+human+bone+marrow+stromal+cells+differentiate+into+neurons.">Adult rat and human bone marrow stromal cells differentiate into neurons.</a> J Neurosci Res. 61 (4), 364-370 (2000).</li><li>Sakaguchi, Y., Sekiya, I., Yagishita, K., Muneta, T. <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+human+stem+cells+derived+from+various+mesenchymal+tissues:+Superiority+of+synovium+as+a+cell+source.">Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source.</a> Arthritis Rheum. 52 (8), 2521-2529 (2005).</li><li>De Coppi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+fluid+and+bone+marrow+derived+mesenchymal+stem+cells+can+be+converted+to+smooth+muscle+cells+in+the+cryo-injured+rat+bladder+and+prevent+compensatory+hypertrophy+of+surviving+smooth+muscle+cells.">Amniotic fluid and bone marrow derived mesenchymal stem cells can be converted to smooth muscle cells in the cryo-injured rat bladder and prevent compensatory hypertrophy of surviving smooth muscle cells.</a> J Urol. 177 (1), 369-376 (2007).</li><li>Arufe, M. C., De la Fuente, A., Fuentes, I., de Toro, F. J., Blanco, F. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 Rheum. 58 (6), 1731-1740 (2008).</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 (Oxford). 47 (8), 1137-1143 (2008).</li><li>Sekiya, I., 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+mesenchymal+stem+cells+in+synovial+fluid+increase+in+the+knee+with+degenerated+cartilage+and+osteoarthritis.">Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis.</a> J Orthop Res. 30 (6), 943-949 (2011).</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>Cooper, D. K., Gollackner, B., Sachs, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Will+the+pig+solve+the+transplantation+backlog.">Will the pig solve the transplantation backlog.</a> Annu Rev Med. 53, 133-147 (2002).</li><li>Millard, A. L., Mueller, N. 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W., Niemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perspectives+for+porcine-to-human+xenografts.">The perspectives for porcine-to-human xenografts.</a> Comp Immunol Micro biol Infect Dis. 32 (2), 91-105 (2009).</li><li>Zeng, L., 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=Multipotent+adult+progenitor+cells+from+swine+bone+marrow.">Multipotent adult progenitor cells from swine bone marrow.</a> Stem Cells. 24 (11), 2355-2366 (2006).</li><li>Zeng, L., 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=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Sekiya, I., 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+mesenchymal+stem+cells+in+synovial+fluid+increase+in+the+knee+with+degenerated+cartilage+and+osteoarthritis.">Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis.</a> J Orthop Res. 30 (6), 943-949 (2012).</li><li>Mochizuki, 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=Higher+chondrogenic+potential+of+fibrous+synovium-+and+adipose+synovium-derived+cells+compared+with+subcutaneous+fat-derived+cells:+Distinguishing+properties+of+mesenchymal+stem+cells+in+humans.">Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: Distinguishing properties of mesenchymal stem cells in humans.</a> Arthritis Rheum. 54 (3), 843-853 (2006).</li></ol>

Miniporcos foram utilizados para estabelecer o isolamento MSCs a partir de medula óssea e no fluido sinovial. Para eliminar várias condições fisiológicas, tais como idade, sexo, e as doenças, minipigs de isogï¿½nicas doadores fundo foram escolhidos para avaliar com precisão a caracterização depende fonte de células. Os porcos são conhecidos por serem anatomicamente, fisiologicamente, e geneticamente semelhantes aos seres humanos, e em particular, podem produzir miniporcos órgãos pareados por tamanho; por...

Multipotent mesenchymal stem cells (MSCs) can be classified into mesenchymal cell lineages, and MSCs have been established and isolated from various tissue sources, such as from bone marrow, umbilical cords, placentas, adipose tissue, dermal skin, skeletal muscle, hair follicles, synovial membranes, and teeth1-5. Currently, attention has been given to synovial-derived MSCs because these cells may help treat joint diseases, such as bone fraction, osteoarthritis, and rheumatoid arthritis (RA), and due to the regenerative potential of MSCs in damaged cartilage or bone, they may also help treat immune modulation or autoimmune diseases6-8. The majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid2,9; thus, the biological understanding of synovial-fluid-derived MSCs is limited.
Synovial fluid is an easily accessible source of MSCs, which can be aspirated during the diagnoses or treatments of patients and during diagnostic confirmations of various arthritis conditions that do not require invasive procedures, such as osteoarthritis or RA. The population of synovial-fluid-derived MSCs in the synovial cavity is significantly increased in arthritic patients compared to non-arthritic patients10-12. Therefore, synovial fluid is an excellent source candidate for MSCs, particularly for autologous stem cell therapy in patients with inflamed or injured joints. Additionally, synovial-fluid-derived MSCs have a chondrogenic capacity, immunomodulation abilities, and a high rate of proliferation. This study established protocols for bone marrow extraction and synovial fluid aspiration from minipigs using non-invasive methods and for the in vitro expansion of isolated cells and analysis of cell phenotypes.

<table border="0" cellpadding="0" cellspacing="0" width="499">
	<tbody>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">Advanced Dulbecco&#8217;s modified Eagle medium (ADMEM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">12491-023</td>
			<td style="width:143px;">MSC culture medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">14190-144</td>
			<td style="width:143px;">Cell washing medium, free of Ca2+/Mg2+,</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:136px;">Fetal bovine serum (FBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">16000-044</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">35050-061</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Penicillin/streptomycin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">10378-016</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:143px;">Simga</td>
			<td style="width:79px;">F0291</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Bovine serum albumin (BSA)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">A6003</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Trypsin-EDTA</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">25200-072</td>
			<td style="width:143px;">Cell dissociation reagent&#160;</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">&#946;-mercaptoethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">M7522</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Isotype antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 550616</td>
			<td style="width:143px;">Isotype Control</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:136px;">CD29 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 552369</td>
			<td style="width:143px;">Integrin beta-1 MSCs marker</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">CD44 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 553133</td>
			<td style="width:143px;">Cell surface glycoprotein MSCs marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD45 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 340664</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD34 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555821</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">CD90 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555595</td>
			<td style="width:143px;">Thy-1 membrane glycoprotein MSCs marker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:136px;">MHC Class II antibody</td>
			<td style="width:143px;">Santa Cruz</td>
			<td style="width:79px;">SC-32247</td>
			<td style="width:143px;">Antigen presenting cells marker</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Vimentin antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">Sigma-S6389</td>
			<td style="width:143px;">Type III intermediate filament MSCs marker</td>
		</tr>
		<tr height="115">
			<td height="115" style="height:115px;width:136px;">Alkaline phosphatase</td>
			<td style="width:143px;">Promega</td>
			<td style="width:79px;">S3841</td>
			<td style="width:143px;">Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Ficoll-Paque</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:79px;">17-1440-02</td>
			<td style="width:143px;">Density gradient centrifugation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Cell strainer</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">352340</td>
			<td style="width:143px;">40&#181;m nylon cell strainer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">15-mL polystyrene conical tube</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">351095</td>
			<td style="width:143px;">Sample collection and cell isolation</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:136px;">35 mm dishes</td>
			<td style="width:143px;">Nunc</td>
			<td style="width:79px;">153066</td>
			<td style="width:143px;">Cell culture dish</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:136px;">Bone marrow extractor</td>
			<td style="width:143px;">GmbH Medizintechnologie, Germany</td>
			<td style="width:79px;">1145-1W010</td>
			<td style="width:143px;">TrokaBone, 3.0x100mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Hematocritchamber</td>
			<td style="width:143px;">Marinfeld</td>
			<td style="width:79px;">640130</td>
			<td style="width:143px;">Cell counting chamber</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:136px;">Atropine</td>
			<td style="width:143px;">Jeil Pharmaceutical Co, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Atropine sulfate Hydrate</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:136px;">Acepromazine</td>
			<td style="width:143px;">Samu Median, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Pre-anaesthetic sedative and antiemetic drug</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Medetomidine</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anesthetic and analgesic drug</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:136px;">Enrofloxacin</td>
			<td style="width:143px;">Bayer Healthcare, Germany</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-biotics</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Meloxicam</td>
			<td style="width:143px;">Over Veterinary Medicine, Argentina</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-inflammatory and analgesic drug</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Isoflurane</td>
			<td style="width:143px;">Hana pharm, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Inhalational anesthesia</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:136px;">Povidone iodine&#160;</td>
			<td style="width:143px;">Korea Pharma, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Ethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">E7023</td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Heparin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">H3393</td>
			<td style="width:143px;">Anti-coagulating agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Formaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">F8775</td>
			<td style="width:143px;">Cell fixation agent</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:136px;">Ophthalmologic ointment&#160;</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Oxytetracycline HCL with polymyxin B sulfate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Circulating water blanket</td>
			<td style="width:143px;">Gaymar Industries</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Warming system during and after anesthesia</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:136px;">Skin stapler</td>
			<td style="width:143px;">Covidien, USA</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Suture skin closure</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">CO2 Incubator</td>
			<td style="width:143px;">Thermo Forma</td>
			<td style="width:79px;">3111</td>
			<td style="width:143px;">Cell culture Equipment</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:136px;">Flow cytometer</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Cell analyzer</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:136px;">Minipig</td>
			<td style="width:143px;">PWG Genetics Korea, Ltd.</td>
			<td style="width:79px;">T-type</td>
			<td style="width:143px;">Miniature pig</td>
		</tr>
	</tbody>
</table>

isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

Estabelecimento de MSCs derivadas da medula óssea e do fluido sinovial de miniporcos MSCs foram isolados com sucesso a partir da medula óssea e das articulações sinoviais articulares dos porquinhos e expandidas in vitro (Figura 2). Seringa de aspiração de fluido sinovial é simples, e é possível obter células aderentes suficientes durante a cultura in vitro das célu...

Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Isolation and Cellular fenotipagem de células-tronco mesenquimais derivadas do líquido sinovial e Medula Óssea de miniporcos

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

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