Name: Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model (Video) | JoVE Journal
Uploaded: 2018-08-10T14:00:00.000+00:00
Duration: 10 min 20 s
Description: Learn magnetic-activated cell sorting to isolate mesenchymal stem cells from rabbit synovial fluid. This simple, economic protocol improves purification efficiency for regenerative medicine research.

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5 minutes

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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<li>Makker, K., Agarwal, A., Sharma, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+activated+cell+sorting+%28MACS%29%3A+utility+in+assisted+reproduction%5BTitle%5D)+AND+%22Indian+Journal+of+Experimental+Biology%22%5BJournal%5D)">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/pubmed?term=((Magnetic+activated+cell+sorting+%28MACS%29+%E2%80%94+a+new+immunomagnetic+method+for+megakaryocytic+cell+isolation%3A+comparison+of+different+separation+techniques%5BTitle%5D)+AND+%22European+Journal+of+Haematology%22%5BJournal%5D)">Magnetic activated cell sorting (MACS) — 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/pubmed?term=((Devices+for+stem+cell+isolation+and+delivery%3A+current+need+for+drug+discovery+and+cell+therapy%5BTitle%5D)+AND+%22Expert+Review+of+Medical+Devices%22%5BJournal%5D)">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. A method to isolate and purify human bone marrow stromal stem cells. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGronthos%2C+S+%22Mesenchymal+Stem+Cells%22">Mesenchymal Stem Cells</a>. Prockop, D. J., Bunnell, B. A., Phinney, D. G. , Vol. 449 of Methods in Molecular Biology 45-57 (2008).</li>
<li>Krawetz, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synovial+fluid+progenitors+expressing+CD90%2B+from+normal+but+not+osteoarthritic+joints+undergo+chondrogenic+differentiation+without+micro-mass+culture%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">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/pubmed?term=((Purified+human+synovium+mesenchymal+stem+cells+as+a+good+resource+for+cartilage+regeneration%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">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/pubmed?term=((Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells%3A+the+International+Society+for+Cellular+Therapy+position+statement%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">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/pubmed?term=((Modulating+chondrocyte+hypertrophy+in+growth+plate+and+osteoarthritic+cartilage%5BTitle%5D)+AND+%22Journal+of+Musculoskeletal+%26+Neuronal+Interact%22%5BJournal%5D)">Modulating chondrocyte hypertrophy in growth plate and osteoarthritic cartilage.</a> Journal of Musculoskeletal & Neuronal Interact. 8 (4), 308(2008).</li>
<li>Cooke, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+chondrocytes+promotes+chondrogenic+differentiation+without+hypertrophy%5BTitle%5D)+AND+%22Osteoarthritis+and+Cartilage%22%5BJournal%5D)">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/pubmed?term=((Human+articular+chondrocytes+secrete+parathyroid+hormone-related+protein+and+inhibit+hypertrophy+of+mesenchymal+stem+cells+in+coculture+during+chondrogenesis%5BTitle%5D)+AND+%22Arthritis+and+Rheumatism%22%5BJournal%5D)">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

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' isolation, purification, and characterization and the in vitro culture protocol is shown in Figure 1.
Cell Morphology after Magnetic Activated Cell Sorting (MACS) with CD...

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 ...

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Research

JoVE Journal

Biology

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.

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

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.

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

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.

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

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.

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

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

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.

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

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

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

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

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.

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

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.

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

This protocol describes a magnetic bead-based sorting method using phycoerythrin (PE)-labeled flow cytometry antibodies and anti-PE microbeads to isolate specific mesenchymal stem cell subpopulations with high postsort target-marker positivity and preserved CCK-8-based proliferation/metabolic activity under the tested conditions, without requiring expensive cell sorters or target-specific custom beads.

Isolation and Validation of Podoplanin-positive Mesenchymal Stem Cell Subpopulations Using an Indirect Magnetic Bead Strategy

Learn magnetic-activated cell sorting to isolate mesenchymal stem cells from rabbit synovial fluid. This simple, economic protocol improves purification efficiency for regenerative medicine research.

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model (Video) | JoVE Journal

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

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