For chondrogenesis, the EBs were cultured for 5 days, trypsinized into single cells and counted. Next, three drops of 15- µL medium each containing 3×105 cells were pipetted onto the center of the scaffolds, which were placed in a 24-well plate. The seeded cells were allowed to attach for 2 h, and then each well was supplemented with 0.5-mL chondrogenesis differentiation medium (Invitrogen) containing high-glucose DMEM with 10% FBS, 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 50 µmol/mL ascorbic acid, 100 nmol/L dexamethasone and 10 ng/mL TGF-β1, according to the manufacturer's instructions. Identical numbers of of cells were cultured directly in the wells as a control. The medium was changed every 2 days and the cells were collected at 2 and 3 weeks for further analysis.
Chondrogenesis differentiation medium
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Chondrogenesis differentiation medium is a culture medium designed to support the differentiation of mesenchymal stem cells (MSCs) into chondrocytes, the cells responsible for the formation of cartilage. The medium provides the necessary components to promote the chondrogenic lineage commitment and maturation of MSCs.
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Lab products found in correlation
Osteogenesis differentiation medium, by Thermo Fisher Scientific (1 mentions)
Adipogenesis differentiation medium, by Thermo Fisher Scientific (1 mentions)
Alizarin red staining, by Thermo Fisher Scientific (1 mentions)
Adipogenesis assay kit, by Cayman Chemical (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Alcian blue solution, by Merck Group (1 mentions)
5 protocols using chondrogenesis differentiation medium
1
Chondrogenic Differentiation in 3D Scaffolds
2
Quantitative Analysis of Stem Cell Differentiation
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For assays of adipogenesis or osteogenesis, expanded single cells during passages three to five were seeded at the density of 2.5 × 104 cells/cm2 in 24-well plates in DMEM with 10% FBS. At 90% confluence, the medium was switched to the adipogenesis differentiation medium or the osteogenesis differentiation medium, respectively (Invitrogen) and changed every 3 days. After 21 days of culturing, cells were fixed with 4% formaldehyde and stained with oil red O for adipocytes by adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) or with 2% Alizarin Red for osteocytes following the manufacturer's protocol. Cells with oil droplet stained by Oil Red were quantified by measuring at OD at 492 nm in triplicate cultures. Mineralized cells with positive Alizarin Red staining (Alfa Aesar, Tewksbury, MA, USA) were quantified by measuring OD at 405 nm in triplicate cultures.
For the chondrogenesis assay, pellets were prepared by spinning down 1 × 105 cells and incubating in a 15-mL conical tube in chondrogenesis differentiation medium (Invitrogen) with the medium changed every 3 days. After 28 days of culturing, cells were fixed with 4% formaldehyde, and stained with Alcian Blue (Merck, Darmstadt, Germany).
For the chondrogenesis assay, pellets were prepared by spinning down 1 × 105 cells and incubating in a 15-mL conical tube in chondrogenesis differentiation medium (Invitrogen) with the medium changed every 3 days. After 28 days of culturing, cells were fixed with 4% formaldehyde, and stained with Alcian Blue (Merck, Darmstadt, Germany).
3
Chondrogenesis Differentiation of rMDSC Pellets
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rMDSC and rCh pellets were established in microcentrifuge tubes by suspension of 2 × 105 cells in 400 µl of the medium. The tubes were centrifuged at x 300 g (4°C) for 6 min. The tops of the tubes were perforated with an 18-gauge needle after centrifugation to permit gaseous exchange. After 72 h, corresponding to the first time, the medium was changed, and the pellets were gently detached from the bottom. This procedure was repeated every 3 days until the 21-day pellet-culturing time-point was reached. The rMDSC pellet was cultured in a chondrogenesis differentiation medium (Gibco, United Kingdom). Control pellets containing rMDSC cells were cultured using an identical cell culture medium described in Section 2.5 . The triplets of both pellets including rMDSC and control were formed.
4
Chondrocyte Differentiation Assay
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Cartilage differentiation was induced by Chondrogenesis Differentiation Medium (Gibco). Micromass pellet of cells was generating in the center of well plate. After 1 hour of incubation in 37°C the Differentiation Medium was added and culture was continued for additional 14 days with medium replaced every 2-3 days. Subsequently cells were fixed with 4% PFA for 30 minutes and washed with PBS.
To demonstrate positive effect of differentiation into chondrocytes cells were stained with 1% Alcian Blue solution (Sigma-Aldrich). Fixed WJ-MSC or AD-MSC were stained with prepared solution for 30 minutes and washed three times with 0.1 N HCl. At the end, to neutralize acidity the cells were rinsed with distilled water.
To demonstrate positive effect of differentiation into chondrocytes cells were stained with 1% Alcian Blue solution (Sigma-Aldrich). Fixed WJ-MSC or AD-MSC were stained with prepared solution for 30 minutes and washed three times with 0.1 N HCl. At the end, to neutralize acidity the cells were rinsed with distilled water.
5
Chondrogenic Pellet Culture Protocol
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hMDSC and chondrocyte (hCH) pellets were established in microcentrifuge tubes by suspension of 2 × 105 cells in 400 µL of culture medium. The tubes were centrifuged at 300× g (4 °C) for 6 min. The tops of the tubes were perforated with 18-gauge needle after centrifugation to permit gaseous exchange. After 72 h, corresponding to the first time the medium was changed, the pellets were gently detached from the bottom. This procedure was repeated every 3 days until the 21 days pellet culturing time point was reached. The hMDSCs pellet was cultured in a chondrogenesis differentiation medium (Gibco, UK). Control pellet containing hMDSC cells were cultured using an identical cell culture medium described in the section of hMDSC isolation and monoculture. The triplets of both pellets including hMDSC and control were formed.
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