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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 tissue^2,3,4. Findings have also confirmed the presence of MSCs in synovial fluid, and previous research has identified synovial fluid-derived MSCs (SF-MSCs) as promising candidates for articular regeneration^5,6.

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 Therapy^8,9. In particular, synovial fluid chondroprogenitors are capable of non-hypertrophic chondrogenesis when induced by TGF-β1, thus making them suitable cell sources for phenotypically articular cartilage regeneration^10,11,12.

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.

Protokół

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's Hospital, Shenzhen University.

1. Isolate and Culture the rbSF-MSCs

1. Preparations for the animal procedure
   1. 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 °C for body temperature.
   2. Fast the animals for 6 h before anesthesia.
2. Preparations and procedure for the animal anesthesia
   1. 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.
   2. Place the rabbit in a comfortable dorsal-recumbent position.
   3. Monitor the respiratory rate, heart rate, and body temperature of the rabbit during anesthesia.
   4. Use ophthalmologic ointment on its eyes to prevent dryness during anesthesia.
   5. Assess the depth of anesthesia following Guedel's classification¹⁴.
3. Preparation for MSCs isolation and cultivation
   1. 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.
   2. Incubate the culture medium at 37 °C in a water bath.
   3. Prepare 500 mL of phosphate buffer saline (PBS) for cell isolation and cell washing.
   4. Prepare 100 mL of isotonic saline solution for the knee cavity arthrocentesis procedure.
4. Collection of articular synovial fl uid from the knee of the rabbit
   1. 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.
   2. Alternately, disinfect the procedure site 3x with povidone iodine solution and 75% ethanol. Then, apply sterile drapes after the area has thoroughly dried.
   3. 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.
   4. Filter the synovial fluid through a 40 µm nylon cell strainer to remove any debris within 4 h.
5. Culture of the rbSF-MSCs
   1. Collect the filtered fluid in 50 mL centrifuge tubes and centrifuge at 1,500 rpm for 10 min at room temperature.
   2. 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.
   3. Incubate the dishes at 37 °C in a humidified atmosphere containing 5% CO₂.
   4. 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.
   5. 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.
   6. Discard the colonies < 2 mm wide, using cell scrapers. Digest the selected colonies with about 5 µL of 0.25% trypsin, using a cloning cylinder, and transfer the colonies to a new dish as passage 1 (P1).
6. Post-operative animal care
   1. Follow standard institutional operating procedures for post-operative monitoring and recovery from anesthesia.
   2. 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.
   3. Post-operation, disinfect the site with 0.1% povidone iodine, 2x a day for 3 days.

2. CD90-positive Magnetic Activated Cell Sorting (MACS) of the rbSF-MSCs and Primary Culture

1. Sample preparation
   1. When the cells reach around 80% confluency, aspirate the medium, and then add 1 - 2 mL of 0.25% Trypsin-EDTA to each dish.
   2. Incubate the dishes for 2 - 3 min to allow cell detachment.
   3. Once the cells are detached, add an equal amount of the culture medium to inactivate the trypsin.
   4. Pass the cell suspension through a 40 µm cell strainer, collect the filtrate in a 15 mL tube, and then spin down the cells at 600 × g for 10 min at room temperature.
   5. 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.
2. Magnetic labeling
   NOTE: Sort the rbSF-MSCs with a magnetic-activated cell sorting kit containing columns, a stand, and separators (see Table of Materials).
   1. Determine the cell number using a hemocytometer.
   2. Centrifuge the cell suspension at 300 × g for 10 min at 4 °C. Completely aspirate the supernatant.
   3. Add 80 µL of resuspension buffer per 10⁷ total cells.
   4. For 10⁷ total cells, add 20 µL of microbeads conjugated with a monoclonal anti-rabbit CD90 antibody.
   5. Mix the magnetic beads and cells evenly in the tubes, then incubate them at 4 °C for 15 min in the dark.
   6. Add 1 mL of buffer per 10⁷ cells to the tube, and then centrifuge it at 300 × g for 10 min at 4 °C to wash the cells. Discard the supernatant after the centrifugation.
   7. Resuspend the pellet in 500 µL of buffer per 10⁷ cells.
3. Magnetic separation
   1. Place the column with the column wings to the front into the magnetic field of the magnetic separator.
   2. Rinse the magnetic separator (MS) column with 500 µL of the buffer per 10⁷ cells.
   3. Transfer the single cell suspension into the column. Let the negative cells pass through the magnetic field to discard these unlabeled cells.
   4. Wash the column 1 - 2x with 500 µL of the buffer per 10⁷ cells and discard the flow-through.
   5. Transfer the column into a centrifuge tube (15 mL).
   6. 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.
   7. Repeat the aforementioned procedure with a second MS Column to increase the purity of CD90⁺ cells, which can enrich the eluted fraction.
   8. Centrifuge the cell suspension at 300 × g for 10 min, aspirate the supernatant, and resuspend it with the culture medium.
4. Culture of the CD90⁺ rbSF-MSCs
   1. Inoculate the cells in 100 mm dishes after the magnetic activated cell sorting.
   2. Incubate the dishes at 37 °C in a humidified cell incubator with 5% CO₂.
   3. 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).
   4. 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.

3. Identification of rbSF-MSCs

1. Surface marker confirmation of the rbSF-MSCs by flow cytometry
   1. 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 °C for 2 - 3 min until the cells are detached.
   2. Harvest the cells using 10 mL of PBS, transfer them into a conical tube (15 mL), and centrifuge them at 300 × g for 5 min at room temperature.
   3. Discard the supernatants. Resuspend the cell pellet in 500 µL of PBS and transfer it into a 1.5 mL tube.
   4. 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 °C.
   5. Centrifuge this mixture at 300 × g for 5 min at room temperature and wash the cells twice with PBS by centrifugation at 300 × g for 5 min each time. Discard the supernatants.
   6. Resuspend the cells in 500 µL 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.
2. Multidifferentiation of the rbSF-MSCs
   NOTE: Use P3 rbSF-MSCs for the in vitro multidifferentiation assays.
   1. Osteogenic differentiation
      1. Prepare the osteogenic induction medium: DMEM basic (1x) containing 50 mM of L-ascorbic acid-2-phosphate,10 mM of α-glycerophosphate, and 100 nM of dexamethasone.
      2. Seed the cells at 10³ cells/cm² in a 6-well tissue culture plate and culture them in the osteogenic induction medium.
      3. Change the induction medium every 3 days for 3 weeks.
      4. 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.
   2. Adipogenic differentiation
      1. 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.
      2. Treat the P3 rbSF-MSCs for 3 weeks in the adipogenic induction medium.
      3. 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.
   3. Chondrogenic differentiation
      1. Prepare the chondrogenic induction medium: DMEM basic (1x) consisting of 10 ng/mL of TGFβ1, 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.
      2. Use pellet culturing for the chondrogenic induction. Centrifuge 5×10⁵ P3 rbSF-MSCs at 1500 rpm for 10 min in a polypropylene tube (15 mL) to form a pellet.
      3. Culture the pellets for 3 weeks with the chondrogenic induction medium.
      4. Change the medium every 3 days for 3 weeks.
      5. 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.
3. Total RNA extraction and analysis by quantitative real-time polymerase chain reaction (qRT-PCR)
   1. Isolate the total RNA of the cells after 3 weeks' differentiation induction using the commercial RNA extraction kit (see Table of Materials).
      1. Remove the culture medium in the culture dish and then add 1 mL of isolation reagent.
      2. Lyse the cells by repeated pipetting until the solution is homogeneous. Incubate it at room temperature for 3 min.
      3. Transfer the cell lysate into a 1.5 mL microcentrifuge tube.
      4. Add 100 µL of chloroform, shake it by hand 15x, and incubate the mixture at room temperature for 3 min.
      5. Centrifuge the tube at 12,000 × g for 15 min at 4 °C. Transfer the supernatants into a new 1.5 mL microcentrifuge tube.
      6. Add 500 µL of isopropanol and precipitate the RNA for 10 min at room temperature.
      7. Centrifuge at 12,000 × g for 10 minat 4 °C. The RNA pellet should be visible at the bottom of the tube.
      8. Remove the supernatant and then add 500 µL of 75% ethanol. Briefly spin the tube for several seconds to wash the RNA pellet. Centrifuge it at 7,500 × g for 5 min at 4 °C.
      9. Remove the residual ethanol.
      10. Dissolve the RNA pellet in 10 µL of DEPC water and place the tubes on ice.
   2. Reverse transcribe total RNA into cDNA with a DNA synthesis kit (see Table of Materials).
      NOTE: Perform all the following procedures on ice.
      1. Prepare the reverse transcription master mix.
      2. Add 25 µL of DNase-treated RNA to each tube with 1 µg of RNA.
      3. Incubate the mixture at the following temperatures using a PCR thermal cycler: 26 °C for 10 min (to allow the random hexamers to anneal), 42 °C for 45 min (reverse transcription) and 75 °C for 10 min (to inactivate the reverse transcriptase).
      4. Immediately analyze the resulting cDNA by quantitative real-time PCR, or store it at -20 °C.
   3. Perform the gene expression analysis with a quantitative real-time PCR system.
      1. Use qRT-PCR Master Mix (see Table of Materials) to perform PCR. The PCR primers for GAPDH, Runx2, Agg, and PPARγ are listed in Table 1.
      2. Calculate gene expression using the 2^−ΔΔCT method.
      3. Conduct a statistical analysis using standard statistical software. Use an independent sample t-test to compare the group means.

Wyniki

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) w...

Dyskusje

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 injury^18,19. The clinical application of SF-MSCs has rarely been cov...

Materiały

Name	Company	Catalog Number	Comments
Reagents
MesenGro	StemRD	MGro-500 1703	Warm in 37 °C water bath before use
MesenGro Supplement	StemRD	MGro-500 M1512	Component of MSCs culture medium
DMEM basic	Gibco Inc.	C11995500BT	MSCs differentiation medium
Isotonic saline solution	Litai, China	5217080305	Cavity arthrocentesis procedure reagent
Phosphate-Buffered Saline (PBS)	HyClone Inc.	SH30256.01B	PBS, free of Ca2+/Mg2+
Fetal Bovine Serum (FBS)	Gibco Inc.	10099-141	Component of MSCs culture medium
Povidone iodine solution	Guangdong, China	150605	Sterilization agent
75% ethanol	Lircon, china	170917	Sterilization agent
0.25% Trypsin/EDTA	Gibco Inc.	25200-056	Cell dissociation reagent
1% Penicillin-Streptomycin	Gibco Inc.	15140-122	Component of MSCs medium
MACS Running Buffer	MiltenyiBiotec	5160112089	Containing phosphate-buffered saline (PBS), 0.5% bovine serum albumin(BSA), and 2 mMEDTA
CD90 antibody conjugated MicroBeads	MiltenyiBiotec	5160801456	For magnetic activated cell sorting
Sodium pyruvate	Sigma-Aldrich	P2256	Component of MSCs chondrogenic differentiation
Dexamethasone	Sigma-Aldrich	D1756	Component of MSCs osteogenic differentiation
ITS	BD	354352	1%, Component of MSCs chondrogenic differentiation
L-proline	Sigma-Aldrich	P5607	0.35 mM, Component of MSCs chondrogenic differentiation
L-ascorbic acid-2-phosphate	Sigma-Aldrich	A8960	50 mM, Component of MSCs chondrogenic differentiation
3-isobutyl-1-methylxanthine	Sigma-Aldrich	I5879	0.5 mM, Component of adipogenic differentiation
Indomethacin	Sigma-Aldrich	I7378	100 mM, Component of adipogenic differentiation
TGFβ1	Peprotech	100-21	10 ng/mL, Component of MSCs chondrogenic differentiation
α-glycerophsphate	Sigma-Aldrich	G6751	Component of MSCs osteogenic differentiation
CD34 Polyclonal Antibody, FITC Conjugated	Bioss	bs-0646R-FITC	Hematopoietic stem cells marker
Mouse antirabbit CD44	Bio-Rad	MCA806GA	Thy-1 membrane glycoprotein (MSCs marker)
CD45 (Monoclonal Antibody)	Bio-Rad	MCA808GA	Hematopoietic stem cells marker
CD105 antibody	Genetex	GTX11415	MSCs marker
Isopropyl alcohol	Sigma-Aldrich	I9030	Precipitates RNA extraction organic phases
Trichloromethane	Wenge, China	61553	Extract total RNA
Trizol	Invitrogen	15596-018	Isolate total RNA
SYBR green master mix	Takara Bio, Japan	RR420A	PCR test
cDNA synthesis kit	Takara Bio, Japan	RR047A	Reverse-transcribed to complementary DNA
Alizarin Red	Sigma-Aldrich	A5533	Staining of calcium compounds
Toluidine Blue	Sigma-Aldrich	89640	Staining of cartilaginous tissue
Oil Red O solution	Sigma-Aldrich	O1391L	Lipid vacuole staining
Equipment
MiniMACS Separator	MiltenyiBiotec	130-042-102	For magnetic activated cell sorting
MultiStand	MiltenyiBiotec	130-042-303	For magnetic activated cell sorting
MS Columns	MiltenyiBiotec	130-042-201	For magnetic activated cell sorting
Cell Strainer	FALCON Inc.	352340	40 μm nylon
Hemocytometer	ISOLAB Inc.	075.03.001	Cell counting
Falcon 100 mm dish	Corning	353003	Cell culture dish
Microcentrifuge tube	Axygen	MCT-150-C	RNA Extraction and PCR
Centrifuge Tubes	Sigma-Aldrich	91050	Gamma-sterilized
High-speed centrifuge	Eppendorf	5804R	Centrifuge cells
Carbon dioxide cell incubator	Thermo scientific	3111	Cell culture
Real-Time PCR Instrument	Life Tech	QuantStudio	Real-Time quantitative polymerase chain reaction
Flow cytometer	BD Biosciences	342975	Cell analyzer
Pipettor	Eppendorf	O25456F	Transfer the liquid
Cloning cylinder	Sigma-Aldrich	C3983-50EA	Isolate and pick individual cell colonies
Sterile hypodermic syringe	Double-Dove, China	131010	Arthrocentesis procedure
Rabbit cage	Zhike, China	ZC-TGD	Restrain the rabbit