A co-culture system was established using six-well Transwell plates (Corning, USA). 3 × 105 BMSCs were cultured in the upper compartments and 3 × 105 SnChos, Chos, or SnChos that had been pretreated by ABT-263 were cultured in the lower compartments in DMEM with 10% FBS. Chondrogenic differentiation medium (Gibco, USA) was used to culture for MSC stemness and to evaluate differentiation. BMSCs and SnChos alone were cultured as the control. Co-cultures were maintained for 7 and 21 days before evaluation. Cells were passaged every 7 days during the 21-day co-culture. Co-cultures were conducted in technical triplicate for each assay and five random field of each well were selected for evaluation.
Chondrogenic differentiation medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chondrogenic differentiation medium is a cell culture medium designed to support the differentiation of cells towards a chondrocyte (cartilage) lineage. It provides the necessary nutrients and growth factors to promote the development of cartilage tissue in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Adipogenic differentiation medium, by Thermo Fisher Scientific (3 mentions)
Osteogenic differentiation medium, by Thermo Fisher Scientific (3 mentions)
Toluidine blue, by Merck Group (2 mentions)
Oil red o, by Merck Group (2 mentions)
Alcian blue, by Merck Group (2 mentions)
Alizarin red s, by Merck Group (2 mentions)
Transwell plate, by Corning (1 mentions)
Tri lineage differentiation kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Leica (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Conical tube, by BD (1 mentions)
Hematoxylin eosin stain, by Merck Group (1 mentions)
Oil red o dye, by Merck Group (1 mentions)
Mesencult acf medium, by STEMCELL (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometry, by BD (1 mentions)
10 protocols using chondrogenic differentiation medium
1
BMSC-Chondrocyte Co-culture Differentiation
2
Tri-Lineage Differentiation of Mesenchymal Stem Cells
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The differentiation capacity of MSCs was conducted using the Tri-lineage Differentiation Kit (Gibco BRL, Grand Island, NY, USA) as described previously [32 (link)]. For osteogenic differentiation, MSCs were seeded into 24-well plates and cultured with an osteogenic differentiation medium (Gibco) for 21 days. The osteogenic differentiation was confirmed by the appearance of Alizarin Red stain. For adipogenic differentiation, MSCs were cultured in an adipogenic differentiation medium (Gibco) for 7 days. The adipogenic differentiation was confirmed by the cellular accumulation of neutral lipid vacuoles, which were stained red with Oil Red O. For chondrogenic differentiation, MSCs were collected in 15-mL centrifuge tubes and cultured in a chondrogenic differentiation medium (Gibco). After 21 days of differentiation induction, the pellets were sectioned, and then, sulfated proteoglycans were visualized by staining with 1% toluidine blue (Merck, Darmstadt, Germany) for 10 min. The chondrogenic differentiation was confirmed by the appearance of Alcian Blue stain.
3
Chondrogenic Differentiation of MSCs
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The bone marrow-derived MSCs were collected in 15 mL centrifuge tubes containing approximately 2 × 105 per tube and subsequently cultured in chondrogenic differentiation medium (Gibco BRL, Grand Island, NY, USA). The medium was refreshed every three days. After 21 days of differentiation induction, the chondroid pellets were generated and washed with PBS and fixed in 4% paraformaldehyde, embedded with optimum cutting temperature (OCT) embedding material (Leica, Wetzlar, Germany). The pellets were sectioned using a freezing microtome, and subsequently, sulfated proteoglycans were visualized by staining with 1% toluidine blue (Merck, Darmstadt, Germany) for 10 min [27 (link)]. These slices were washed 3 times with PBS and photographed under an inverted microscope. The differentiation was confirmed as the appearance of alcian blue staining.
4
Multilineage Differentiation of Mesenchymal Stem Cells
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These procedures were described in detail in our previous study (9 (link)). Briefly, for adipogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (8x104 cells per well), cultured for 12 h and treated with adipogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 7 days; the medium was refreshed every 3 days. Adherent cells were stained red with 60% Oil Red O (Sigma-Aldrich; Merck KGaA) for 1 min at room temperature. For osteogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (4x104 cells per well), cultured for 12 h and treated with osteogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. Osteogenic differentiation was confirmed by 0.2% Alizarin Red staining for 5 min at room temperature. For chondrogenic differentiation, 2x105 MSCs were collected in 15-ml centrifuge tubes and cultured with chondrogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. The chondroid pellets were sectioned (8 µm) with a freezing microtome. The slices were stained with 1% toluidine blue for 3 min at room temperature and were captured using a light microscope (Olympus Corporation) at a x50 magnification.
5
Multilineage Differentiation Potential of MSCs
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IL10-MSCs and control MSCs were seeded in 24-well culture plate at a density of 1 × 104 cells per well in DMEM medium complemented with 10% fetal bovine serum (FBS) (10099141, Gibco). When the cells grew to 50–70% confluency, medium was replaced with adipose and osteogenic differentiation medium (Gibco, USA) to induce adipogenesis and osteogenesis, respectively. After 21 days, cells were fixed in 4% formaldehyde and stained with Oil Red O (Sigma-Aldrich, USA) and Alizarin Red S (Sigma-Aldrich, USA) to evaluate adipogenic and osteogenic differentiation, respectively. In addition, 2 × 105 cells were centrifuged in a tube at 1200 rpm/min for 5 min, and chondrogenic differentiation medium (Gibco, USA) was added to the pellet after the supernatant was removed. After 21 days, the precipitate was fixed in 4% formaldehyde, dehydrated through serial ethanol dilutions, and then embedded in the optimal cutting temperature compound (OCT). The block was cut into 5-mm-thick sections and stained with Alcian Blue (Sigma-Aldrich, USA) to assess chondrogenic differentiation potential.
6
Chondrogenic Differentiation of C3H10T1/2 Cells
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C3H10T1/2 cells were dissociated using 0.25% trypsin and adjusted to a density of 105 cells/ml. Cell suspension (2 ml/well) was placed into the center of each well on a 6-well plate. After incubation for 24 h at 37℃ with 5% CO2, the medium was replaced by 2 ml chondrogenic differentiation medium (Gibco) containing dexamethasone (100 nmol/L), ascorbate (50 µg/ml), ITS+ Supplement, proline (40 µg/ml), and TGF-β3 (10 ng/ml). The chondrogenic differentiation medium was refreshed every 3 d.
7
Chondrogenic Differentiation of HEASCs
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HEASCs were trypsinized, counted, and centrifuged into cell pellets (1.2 × 105 per pellet) in 15 ml conical tubes (BD Falcon). Chondrogenic differentiation medium (Gibco) was gently overlaid so as to not detach the cell nodules, and the culture was maintained in the Chondrogenic differentiation medium for 3 weeks. Afterwards, the pellets were fixed and frozen in O.C.T. (Tissue-Tek, Sakura, Zoeterwoude, Netherlands). The pellets were sectioned, fixed with 4% polymethanol, stained against Alcian blue stain, and further stained with hematoxylin-eosin stain (Sigma-Aldrich).
8
Chondrogenic Differentiation of ECs
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Briefly, 250,000 cells at passage 3 were suspended in 500 mL EGM-2 medium aliquoted into 10 mL sterile tubes, centrifuged at 300 g for 5 min to form pellets, and incubated overnight. Medium was replaced by chondrogenic differentiation medium (Invitrogen) while control cells were cultured in incomplete differentiation medium. Tops were attached loose to allow gas exchange. Culture medium was exchanged every 3–4 days over 4 weeks.
9
Multilineage Differentiation Evaluation
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To assess adipogenic differentiation, 1 × 103 cells/cm2 were cultured in adipogenic differentiation medium (DMEM/F12 supplemented with 10% FBS, 100 nM dexamethasone, and 200 nM indomethacin). After 21 days, as accumulation of lipid-rich vacuoles is detected within cells, Oil Red O staining was done. Osteogenic differentiation was induced in osteogenic differentiation medium (50 µg/ml ascorbic acid, 10 nM dexamethasone, and 10 mM -glycerophosphate) with 3–5 × 103 cells/cm2. After 21 days, Alizarin Red S staining, as determination of calcium accumulation assessment, was performed. Chondrogenic differentiation was persuaded by chondrogenic differentiation medium (Invitrogen, USA) in density of 3–5 × 103 cells/cm2. After 21 days, chondrogenesis was evaluated using Alcian blue staining. The slides were fixed with 4% paraformaldehyde for 30 min and washed with PBS.
10
Comprehensive Characterization of Human AMSCs
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Human AMSCs from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China) were soaked in MesenCult™-ACF medium (STEMCELL Technologies, CA) supplemented with 2 mM L-glutamine (Thermo Fisher Scientific, Wilmington, DE, USA) and 1% antibiotic antibacterial agent (Thermo Fisher Scientific). The phenotypes of the 3rd to 6th generation of AMSCs were evaluated by flow cytometry analysis (BD Accuri®C6 flow cytometry) after incubation with antibodies against CD29, CD31, CD44, CD45, CD73, CD90, CD105, and HLA-DR (Biolegend, CA, USA). IgG1 served as a negative control. Adipogenic differentiation medium (Invitrogen, Carlsbad, CA, USA), osteogenic differentiation medium (Invitrogen), and chondrogenic differentiation medium (Invitrogen) were used to induce the differentiation of AMSCs. After 14 or 21 days of induction, the cells were stained with Oil Red O dye (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), Alizarin Red S (Sigma-Aldrich), and Alcian Blue (Sigma-Aldrich).
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