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 rabbits one day prior to the anesthesia and arthrocentesis procedure. 
		NOTE: Physical examinations should include weight (2.0 - 2.5 kg), gender (female), and body temperature, respiratory rate, and heart rate. Parameter intervals for clinically healthy rabbits are 30 - 50 min for respiration rate, 220 - 280 min for heart rate, and 38 - 39 &#176;C for body temperature.</li>
		<li>Fast the animals for 6 h before anesthesia.</li>
	</ol>
	</li>
	<li>Preparations and procedure for the animal anesthesia
	<ol>
		<li>Restrain the rabbit with a cage (see Table of Materials), and then inject 3% pentobarbital sodium into the marginal ear vein at a dose of 1 mL/kg for general anesthesia.</li>
		<li>Place the rabbit in a comfortable dorsal-recumbent position.</li>
		<li>Monitor the respiratory rate, heart rate, and body temperature of the rabbit during anesthesia.</li>
		<li>Use ophthalmologic ointment on its eyes to prevent dryness during anesthesia.</li>
		<li>Assess the depth of anesthesia following Guedel&#39;s classification14.</li>
	</ol>
	</li>
	<li>Preparation for MSCs isolation and cultivation
	<ol>
		<li>To cultivate rbSF-MSCs, prepare 500 mL of commercial culture medium (see Table of Materials) supplemented with 10% commercial supplement (see Table of Materials), 10% fetal bovine serum (FBS), and 1% Penicillin-Streptomycin.</li>
		<li>Incubate the culture medium at 37 &#176;C in a water bath.</li>
		<li>Prepare 500 mL of phosphate buffer saline (PBS) for cell isolation and cell washing.</li>
		<li>Prepare 100 mL of isotonic saline solution for the knee cavity arthrocentesis procedure.</li>
	</ol>
	</li>
	<li>Collection of articular synovial &#64258; uid from the knee of the rabbit
	<ol>
		<li>Select an area about 5 x 5 cm in size around the knee and shave the rabbit hair from this area using a safety electric shaver.</li>
		<li>Alternately, disinfect the procedure site 3x with povidone iodine solution and 75% ethanol. Then, apply sterile drapes after the area has thoroughly dried.</li>
		<li>Inject 1 - 2 mL of isotonic saline solution, using a sterile hypodermic syringe (2 mL), into the knee joint cavity from the lateral articular space, move the knee 3 - 4x, and then suck out all the synovial fluid at room temperature.</li>
		<li>Filter the synovial &#64258;uid through a 40 &#181;m nylon cell strainer to remove any debris within 4 h.</li>
	</ol>
	</li>
	<li>Culture of the rbSF-MSCs
	<ol>
		<li>Collect the filtered &#64258;uid in 50 mL centrifuge tubes and centrifuge at 1,500 rpm for 10 min at room temperature.</li>
		<li>Discard the supernatant after the centrifugation, wash the pellet with PBS, resuspend the pellet with the complete culture medium, and then plate the medium in 100 mm dishes.</li>
		<li>Incubate the dishes at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
		<li>After 48 h, replenish the dishes with fresh medium to remove any non-adherent cells. Replace the medium twice a week for 2 weeks as passage 0 (P0). 
		NOTE: There should be about 1 x 104 adherent cells. Viable cells/ml in SF are about 2 x 102/mL.</li>
		<li>After 14 days following the initial plating, many colonies should have formed in the culture dishes. Select the colonies larger than 2 mm in diameter. Mark where the selected colonies are located by tracing their circumference on the dish bottom.</li>
		<li>Discard the colonies &#60; 2 mm wide, using cell scrapers. Digest the selected colonies with about 5 &#181;L of 0.25% trypsin, using a cloning cylinder, and transfer the colonies to a new dish as passage 1 (P1).</li>
	</ol>
	</li>
	<li>Post-operative animal care
	<ol>
		<li>Follow standard institutional operating procedures for post-operative monitoring and recovery from anesthesia.</li>
		<li>Monitor the vital signs of the rabbit every 10 min until it has regained consciousness. Finally, transfer the conscious animal to the cage. Do not return a rabbit that has undergone surgery to the company of other animals until it has fully recovered.</li>
		<li>Post-operation, disinfect the site with 0.1% povidone iodine, 2x a day for 3 days.</li>
	</ol>
	</li>
</ol>
2. CD90-positive Magnetic Activated Cell Sorting (MACS) of the rbSF-MSCs and Primary Culture
<ol>
	<li>Sample preparation
	<ol>
		<li>When the cells reach around 80% confluency, aspirate the medium, and then add 1 - 2 mL of 0.25% Trypsin-EDTA to each dish.</li>
		<li>Incubate the dishes for 2 - 3 min to allow cell detachment.</li>
		<li>Once the cells are detached, add an equal amount of the culture medium to inactivate the trypsin.</li>
		<li>Pass the cell suspension through a 40 &#181;m cell strainer, collect the filtrate in a 15 mL tube, and then spin down the cells at 600 &#215; g for 10 min at room temperature.</li>
		<li>Resuspend the cell pellet in MACS running buffer (PBS, pH 7.2, 0.5% bovine serum albumin, and 2 mM EDTA) and then count the cell number.</li>
	</ol>
	</li>
	<li>Magnetic labeling 
	NOTE: Sort the rbSF-MSCs with a magnetic-activated cell sorting kit containing columns, a stand, and separators (see Table of Materials).
	<ol>
		<li>Determine the cell number using a hemocytometer.</li>
		<li>Centrifuge the cell suspension at 300 &#215; g for 10 min at 4 &#176;C. Completely aspirate the supernatant.</li>
		<li>Add 80 &#181;L of resuspension buffer per 107 total cells.</li>
		<li>For 107 total cells, add 20 &#181;L of microbeads conjugated with a monoclonal anti-rabbit CD90 antibody.</li>
		<li>Mix the magnetic beads and cells evenly in the tubes, then incubate them at 4 &#176;C for 15 min in the dark.</li>
		<li>Add 1 mL of buffer per 107 cells to the tube, and then centrifuge it at 300 &#215; g for 10 min at 4 &#176;C to wash the cells. Discard the supernatant after the centrifugation.</li>
		<li>Resuspend the pellet in 500 &#181;L of buffer per 107 cells.</li>
	</ol>
	</li>
	<li>Magnetic separation
	<ol>
		<li>Place the column with the column wings to the front into the magnetic field of the magnetic separator.</li>
		<li>Rinse the magnetic separator (MS) column with 500 &#181;L of the buffer per 107 cells.</li>
		<li>Transfer the single cell suspension into the column. Let the negative cells pass through the magnetic field to discard these unlabeled cells.</li>
		<li>Wash the column 1 - 2x with 500 &#181;L of the buffer per 107 cells and discard the flow-through.</li>
		<li>Transfer the column into a centrifuge tube (15 mL).</li>
		<li>Add 1 mL of the buffer per 107 cells to the column, and then immediately push the plunger into the column to flush out the magnetically labeled cells.</li>
		<li>Repeat the aforementioned procedure with a second MS Column to increase the purity of CD90+ cells, which can enrich the eluted fraction.</li>
		<li>Centrifuge the cell suspension at 300 &#215; g for 10 min, aspirate the supernatant, and resuspend it with the culture medium.</li>
	</ol>
	</li>
	<li>Culture of the CD90+ rbSF-MSCs
	<ol>
		<li>Inoculate the cells in 100 mm dishes after the magnetic activated cell sorting.</li>
		<li>Incubate the dishes at 37 &#176;C in a humidified cell incubator with 5% CO2.</li>
		<li>When 80 - 90% confluence of the primary culture is achieved, at about 7 - 10 days, digest the adherent cells with 0.25% Trypsin-EDTA and passage them at a 1:2 dilution to make passage 2 (P2).</li>
		<li>Use the same method to pass the cells to passage 3 (P3), which can be used for the in vitro assays. 
		NOTE: After the sub-culture and purification, about 1 x 107 rbSF-MSCs are obtained.</li>
	</ol>
	</li>
</ol>
3. Identification of rbSF-MSCs
<ol>
	<li>Surface marker confirmation of the rbSF-MSCs by flow cytometry
	<ol>
		<li>When the MSCs are 80 - 90% confluent at P3, wash the cells with PBS and treat them with 1 mL of 0.25% Trypsin-EDTA. Then, incubate the MSCs at 37 &#176;C for 2 - 3 min until the cells are detached.</li>
		<li>Harvest the cells using 10 mL of PBS, transfer them into a conical tube (15 mL), and centrifuge them at 300 &#215; g for 5 min at room temperature.</li>
		<li>Discard the supernatants. Resuspend the cell pellet in 500 &#181;L of PBS and transfer it into a 1.5 mL tube.</li>
		<li>Incubate a 1:100 dilution of FITC-conjugated and PE-conjugated antibodies (isotype, FITC-CD34/PE-CD45, FITC-CD105/PE-CD44) for 1 h in the dark at 4 &#176;C.</li>
		<li>Centrifuge this mixture at 300 &#215; g for 5 min at room temperature and wash the cells twice with PBS by centrifugation at 300 &#215; g for 5 min each time. Discard the supernatants.</li>
		<li>Resuspend the cells in 500 &#181;L of PBS, transfer the cell suspension into a round-bottom tube (5 mL) and analyze it using a flow cytometer. 
		NOTE: Acquire data on a cytometer equipped with two fluorescence channels: 533/30 and 585/40. For each isotype fluorescence, use the isotype control to adjust the appropriate laser voltage, and set the minimum intensity required to obtain a fluorescence histogram that displays both left and right edges of the peak. Collect a minimum of 10,000 events for the statistical analyses.</li>
	</ol>
	</li>
	<li>Multidifferentiation of the rbSF-MSCs 
	NOTE: Use P3 rbSF-MSCs for the in vitro multidifferentiation assays.
	<ol>
		<li>Osteogenic differentiation
		<ol>
			<li>Prepare the osteogenic induction medium: DMEM basic (1x) containing 50 mM of L-ascorbic acid-2-phosphate,10 mM of &#945;-glycerophosphate, and 100 nM of dexamethasone.</li>
			<li>Seed the cells at 103 cells/cm2 in a 6-well tissue culture plate and culture them in the osteogenic induction medium.</li>
			<li>Change the induction medium every 3 days for 3 weeks.</li>
			<li>Fix the cells with 4% formaldehyde for 30 min at room temperature after the differentiation is completed, stain the cells with 1% Alizarin red for 5 min, and then wash them 3x with PBS15.</li>
		</ol>
		</li>
		<li>Adipogenic differentiation
		<ol>
			<li>Prepare the adipogenic induction medium: DMEM basic (1x) consisting of 100 mM of indomethacin, 10 mg/mL of recombinant human insulin, 1 mM of dexamethasone, and 0.5 mM of 3-isobutyl-1-methylxanthine.</li>
			<li>Treat the P3 rbSF-MSCs for 3 weeks in the adipogenic induction medium.</li>
			<li>After 3 weeks, stain the neutral lipid vacuoles using Oil Red O to confirm the adipogenic differentiation. Fix the cells in a 4% formaldehyde solution for 30 min at room temperature, and then wash them 3x with PBS16.</li>
		</ol>
		</li>
		<li>Chondrogenic differentiation
		<ol>
			<li>Prepare the chondrogenic induction medium: DMEM basic (1x) consisting of 10 ng/mL of TGF&#946;1, 1% ITS supplement, 0.35 mM of L-proline, 100 nM of dexamethasone, 50 mM of L-ascorbic acid-2-phosphate, and 1 mM of sodium pyruvate.</li>
			<li>Use pellet culturing for the chondrogenic induction. Centrifuge 5&#215;105 P3 rbSF-MSCs at 1500 rpm for 10 min in a polypropylene tube (15 mL) to form a pellet.</li>
			<li>Culture the pellets for 3 weeks with the chondrogenic induction medium.</li>
			<li>Change the medium every 3 days for 3 weeks.</li>
			<li>After 3 weeks induction, embed the cell pellet with paraffin, section it into 4 mm slices, and stain them using 0.1% Toluidine blue for 30 min at room temperature17.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Total RNA extraction and analysis by quantitative real-time polymerase chain reaction (qRT-PCR)
	<ol>
		<li>Isolate the total RNA of the cells after 3 weeks&#39; differentiation induction using the commercial RNA extraction kit (see Table of Materials).
		<ol>
			<li>Remove the culture medium in the culture dish and then add 1 mL of isolation reagent.</li>
			<li>Lyse the cells by repeated pipetting until the solution is homogeneous. Incubate it at room temperature for 3 min.</li>
			<li>Transfer the cell lysate into a 1.5 mL microcentrifuge tube.</li>
			<li>Add 100 &#181;L of chloroform, shake it by hand 15x, and incubate the mixture at room temperature for 3 min.</li>
			<li>Centrifuge the tube at 12,000 &#215; g for 15 min at 4 &#176;C. Transfer the supernatants into a new 1.5 mL microcentrifuge tube.</li>
			<li>Add 500 &#181;L of isopropanol and precipitate the RNA for 10 min at room temperature.</li>
			<li>Centrifuge at 12,000 &#215; g for 10 minat 4 &#176;C. The RNA pellet should be visible at the bottom of the tube.</li>
			<li>Remove the supernatant and then add 500 &#181;L of 75% ethanol. Briefly spin the tube for several seconds to wash the RNA pellet. Centrifuge it at 7,500 &#215; g for 5 min at 4 &#176;C.</li>
			<li>Remove the residual ethanol.</li>
			<li>Dissolve the RNA pellet in 10 &#181;L of DEPC water and place the tubes on ice.</li>
		</ol>
		</li>
		<li>Reverse transcribe total RNA into cDNA with a DNA synthesis kit (see Table of Materials). 
		NOTE: Perform all the following procedures on ice.
		<ol>
			<li>Prepare the reverse transcription master mix.</li>
			<li>Add 25 &#181;L of DNase-treated RNA to each tube with 1 &#181;g of RNA.</li>
			<li>Incubate the mixture at the following temperatures using a PCR thermal cycler: 26 &#176;C for 10 min (to allow the random hexamers to anneal), 42 &#176;C for 45 min (reverse transcription) and 75 &#176;C for 10 min (to inactivate the reverse transcriptase).</li>
			<li>Immediately analyze the resulting cDNA by quantitative real-time PCR, or store it at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Perform the gene expression analysis with a quantitative real-time PCR system.
		<ol>
			<li>Use qRT-PCR Master Mix (see Table of Materials) to perform PCR. The PCR primers for GAPDH, Runx2, Agg, and PPAR&#947; are listed in Table 1.</li>
			<li>Calculate gene expression using the 2&#8722;&#916;&#916;CT method.</li>
			<li>Conduct a statistical analysis using standard statistical software. Use an independent sample t-test to compare the group means.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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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 authors declare that they have no competing financial interests.

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>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MesenGro</td><td>StemRD</td><td>MGro-500 1703</td><td>Warm in 37 &amp;deg;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 Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</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&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>10 ng/mL, Component of MSCs chondrogenic differentiation</td></tr><tr><td>&amp;alpha;-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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;mu;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 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 ...

magnetic-activated-cell-sorting-strategies-to-isolate-purify-synovial

Research

JoVE Journal

Biology

11.1K Views.  Guangzhou Medical University. Dit artikel presenteert een eenvoudige en economische protocol voor het eenvoudig isolatie en zuivering van mesenchymale stamcellen uit Nieuw-Zeeland wit konijn gewrichtsvocht.

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

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Developmental Biology

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Medicine

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioengineering

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.

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.

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

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

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

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

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

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