The effects of the MSC-EVs on synovial MSC chondrogenesis were evaluated by pellet culture, as previously described [26 (link)]. Briefly, 1.5 × 106 synovial MSCs were cultured in a 15-mL polypropylene tube (Becton Dickinson) and centrifuged at 1500 rpm for 10 min to form pellets. The pellets were then cultured with either 2 × 109 particles/mL MSC-EVs or PBS in 400 μL chondrogenesis medium (high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen; Thermo Fisher Scientific) supplemented with 1000 ng/mL BMP-7 (Stryker Biotech, Kalamazoo, MI, USA), 10 ng/mL TGF-β3 (R&D Systems, Minneapolis, MN, USA), 100 nmol/L dexamethasone, 50 ng/mL ascorbate-2-phosphate, 40 mg/mL proline, 100 g/mL pyruvate (Sigma-Aldrich), and 50 mg/mL ITS + Premix [Becton Dickinson]) for 21 days. The pellets were fixed, embedded in paraffin, cut into 5-μm sections, and stained with Toluidine Blue for histological evaluation.
15 ml polypropylene tube
Manufactured by BD
Sourced in United States
The 15-mL polypropylene tube is a laboratory equipment item designed to hold and contain liquid samples or substances. It is made of polypropylene, a common plastic material used in various lab applications. The tube has a volume capacity of 15 milliliters.
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Lab products found in correlation
Dexamethasone (dex), by Merck Group (6 mentions)
Its premix, by BD (4 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (4 mentions)
Pyruvate, by Merck Group (4 mentions)
Ascorbate 2 phosphate, by Merck Group (4 mentions)
Tgf β3, by R&D Systems (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Safranin o, by Merck Group (3 mentions)
Tgf β1, by Merck Group (3 mentions)
Proline, by Merck Group (2 mentions)
Its 1, by Merck Group (2 mentions)
Transforming growth factor β3, by R&D Systems (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Tgfβ3, by BioLegend (1 mentions)
Bmp 2, by Merck Group (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
L proline, by Merck Group (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
Transforming growth factor b3, by R&D Systems (1 mentions)
Oasis hlb cartridge, by Waters Corporation (1 mentions)
17 protocols using 15 ml polypropylene tube
1
Synovial MSC Chondrogenesis Evaluation
2
Chondrogenesis of Cell Pellets
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One hundred thousand cells in the 3-days treated group were placed in a 15-ml polypropylene tube (Becton Dickinson) and centrifuged at 450g for 10 min. The pellets were cultured at 37°C with 5% CO2 in 400 μl chondrogenesis medium that contained 1,000 ng/ml BMP-7 (Stryker Biotech, Hopkinton, MA) in high-glucose DMEM (Invitrogen) supplemented with 10 ng/ml transforming growth factor-β3 (R&D Systems, Minneapolis, MN), 100 nM dexamethasone, 50 ng/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate (Sigma–Aldrich, St. Louis, MO), and 50 mg/ml ITS + Premix (Becton Dickinson). The medium was replaced every 3–4 days for 21 days. For microscopy, the pellets were embedded in paraffin, cut into 5-μm sections, and stained with toluidine blue and type II collagen antibody.16 (link)
3
Chondrocyte Pellet Culture and Characterization
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In pellet culture, 4 × 105 of SW1353 or fibroblast cells were centrifuged (150 g, 5 min) in a 15-ml polypropylene tube (Becton Dickinson) to form a pellet. The pellets were treated with DMEM (1 g/L glucose) containing ITS supplement, 0.2 mm L -ascorbic acid-2-phosphate, 1 mm sodium pyruvate, 0.35 mm L-proline (all Sigma-Aldrich), and 10 ng/ml TGFβ3 (Biolegend, 585802) or BMP2 (sigma H4791). Medium was changed every two or 3 days (Ullah et al., 2012 (link); Futrega et al., 2021 (link)). Alcian blue staining and extraction of aggregates were performed as described (Wehrle et al., 2019 (link)).
In 384-well ULA plate, 3 × 103 cells were seeded to each well with the medium mentioned above. Lentivirus transduction and PI staining were performed as described (Tambe et al., 2019 (link)). RFP or GFP plasmids were obtained from Addgene (#13726 and #13727). Fluorescence images were acquired using an LSM 710 confocal microscope (Zeiss, Germany).
In 384-well ULA plate, 3 × 103 cells were seeded to each well with the medium mentioned above. Lentivirus transduction and PI staining were performed as described (Tambe et al., 2019 (link)). RFP or GFP plasmids were obtained from Addgene (#13726 and #13727). Fluorescence images were acquired using an LSM 710 confocal microscope (Zeiss, Germany).
4
Chondrogenic Differentiation of Passage 3 Cells
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A total of 250,000 passage 3 cells were placed in a 15-mL polypropylene tube (Becton Dickinson) and centrifuged at 450g for 10 minutes. The pellets were cultured in chondrogenesis medium (high-glucose Dulbecco’s modified Eagle’s medium [Invitrogen] supplemented with 500 ng/mL of BMP-2, 10 ng/mL of transforming growth factor–β3 [TGF-β3; R&D Systems], 10−7 M of dexamethasone [Sigma-Aldrich], 50 μg/mL of ascorbic acid-2-phosphate, 40 μg/mL of proline, 100 μg/mL of pyruvate, and 50 mg/mL of ITS+ Premix [Becton Dickinson]).9 (link),23 (link),24 (link)
For the microscopic assessment, the pellets were embedded in paraffin, cut into 4-µm sections, and stained with toluidine blue.28 (link)
For the microscopic assessment, the pellets were embedded in paraffin, cut into 4-µm sections, and stained with toluidine blue.28 (link)
5
Chondrogenic Differentiation of Passage-3 Cells
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250,000 passage-3 cells were placed in a 15 mL polypropylene tube (Becton Dickinson) and centrifuged at 450g for 10 min. The pellets were cultured in chondrogenesis medium (high-glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 500 ng/mL BMP-2, 10 ng/mL TGF β3 (R&D Systems; Minneapolis, MN), 10−7 M dexamethasone (Sigma-Aldrich), 50 μg/mL ascorbate-2-phosphate, 40 μg/mL proline, 100 μg/mL pyruvate, and 50 mg/mL ITS+Premix (Becton Dickinson)). For microscopy, the pellets were embedded in paraffin, cut into 4 μm sections, and stained with Toluidine Blue [16 (link)].
6
Chondrogenic Differentiation of Cells
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First, 250,000 cells were placed in a 15-mL polypropylene tube (Becton Dickinson) and centrifuged at 500g for 10 minutes. The pellet was then cultured in 400 mL chondrogenesis medium containing 1,000 ng/mL BMP-7 (Stryker Biotech Hopkinton, MA), 10 ng/mL transforming growth factor b3 (R&D Systems, Minneapolis, MN), and 100 nM dexamethasone (Sigma-Aldrich, St Louis, MO) in high-glucose Dulbecco modified Eagle medium (Invitrogen) for 21 days. For microscopy, the pellets were embedded in paraffin, cut into 5-mm sections, and stained with safranin-o, toluidine blue, and type II collagen antibody (Daiichi Fine Chemical, Toyama, Japan). 11, 12, 20
7
Isolation and Analysis of MHC-Associated Peptides
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Peptides were extracted from the surface of 1×107 cells of TL-DC or HTL-DC pelleted in a 15-mL polypropylene tube (Falcon, Oxnard, CA, USA) as described by Santin, et al.29 (link) with minor modifications. Briefly, 2 mL of citrate-phosphate buffer was added directly to pelleted cells and incubated for 5 min at room temperature. After centrifugation at 500×g for 3 min, supernatant was collected and further clarified by centrifugation at 1800×g for 5 min at 4℃. Peptides were stored at -80℃ until further processing.
Each MHC-associated peptide eluted from TL and HTL was purified using an Oasis HLB cartridge (Waters, Milford, MA, USA), lyophilized using a Modulspin 40 from BioTron (Seoul, Korea), and sequentially resuspended in 100 µL of 2% CH3CN in water for nLC-ESI-MS-MS analysis. Peptides were stored at -80℃ before use.
Each MHC-associated peptide eluted from TL and HTL was purified using an Oasis HLB cartridge (Waters, Milford, MA, USA), lyophilized using a Modulspin 40 from BioTron (Seoul, Korea), and sequentially resuspended in 100 µL of 2% CH3CN in water for nLC-ESI-MS-MS analysis. Peptides were stored at -80℃ before use.
8
Micromass Chondrogenic Differentiation Protocol
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MSCs were harvested and 6×105 cells were centrifuged to form a pellet on the bottom of a 15-ml polypropylene tube (Falcon). The micro mass was cultured in 500 μl of chondrogenic medium that consisted of 50 μg/ml ascorbic acid 2-phosphate and 1 ng/ml TGF-β1 (Sigma) (15 (link)). After 3 weeks of culture, cell clumps were harvested, embedded in paraffin, cut into 3-μm sections, and stained for glycosaminoglycans using 0.1% safranin O (Sigma).
9
Chondrogenic Differentiation of DFATs
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Induction of chondrogenic differentiation by the pellet culture method was used according to a previous report [17 (link)] with slight modification. Briefly, DFATs at P2 were seeded in a 15-ml poly-propylene tube (BD Falcon) at density of 5 × 105 cells with 2 ml of chondrogenic differentiation induction medium (NH Chondrodiff Medium, Miltenyi Biotec). Cells were centrifuged at 500×g for 10 min to precipitate them and cultured at 37 °C under 5% CO2. The medium was exchanged every 3 days. At 21 days of culture, the medium was removed, and the generated micromass pellets were fixed with 4% PFA for 60 min. After being washed with distilled water, the micromass pellets were photographed using the VB-7000 stereomicroscope (Keyence), and the weight of each micromass pellet was measured using an electronic analytical balance (AEG-45SM, Shimazu, Kyoto, Japan). The samples were then subjected to histological analysis and quantification analysis for glycosaminoglycans. In another experiment, DFAT samples at 0, 7, and 14 days of culture were used for RT-PCR analysis.
10
Stem Cell Lineage Differentiation Assay
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For osteogenic, adipogenic and chondrogenic differentiation, commercially differentiation media kits (StemPro Adipogenesis, Chondrogenesis and Osteogenesis Kits; Invitrogen) were used in accordance with the manufacturer instructions. Briefly, cells were seeded at densities of 1 × 104 (osteocyte) and 3.2 × 104 (adipocyte) cells/cm2; for chondrocyte differentiation, cells were seeded at 5 × 105 cells in a 15 ml polypropylene tube (Falcon, Bedford, Mass., USA). For osteocyte differentiation, cells were fixed in 4% paraformaldehyde, stained with fresh 2% alizarin red solution (pH 4.2) for 5 minutes, and then de-stained in 10 mmol L−1 sodium phosphate containing 10% cetylpyridinium chloride (Sigma Diagnostics, St Louis, MO, USA). The amount of alizarin red stain was quantified by measuring the absorbance of the solution at 562 nm. The calcium concentration was measured with a Sigma calcium kit 587-A (Sigma Diagnostics, St Louis, MO, USA). For adipocyte differentiation, cells were fixed in 4% paraformaldehyde for 15 min, and stained with fresh Oil Red-O solution for 15 minutes. For chondrocyte differentiation, the pellets were embedded in paraffin, cut into 5-μm-thick sections, and stained with 1% toluidine blue. The concentrations of chondroitin sulfate and hyaluronan were quantified as reported previously15 (link).
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