To induce differentiation of MSCs into chondrocytes, 2.5 × 105 cells of MSCs were resuspended in 1mL of pre-warmed NH ChondroDiff Medium from Miltenyi Biotec (Bergisch Gladbach, Germany). The medium was changed every third day and on day 24 the formed nodule was fixed in 3.7% neutral buffered formalin and then embedded in paraffin. Using a microtome, the nodule was cut into 5-μm thick tissue sections. After a deparaffinization step, tissue sections were stained with alcian blue (Merck, Darmstadt, Germany), a dye that stains glucosaminoglycans (an important component of the extracellular matrix produced by chondrocytes). The blue to bluish-green stained parts of tissue sections were evaluated by the microscope Olympus IX71 (Olympus, Hamburg, Germany). Differentiation capacity of MSCs into osteoblasts, adipocytes and chondrocytes was graded semiquantitatively by microscopy on the basis of the stained surface of differentiated MSCs by tissue-specific stainings.
Nh chondrodiff medium
Manufactured by Miltenyi Biotec
Sourced in Germany
NH ChondroDiff Medium is a cell culture medium designed for the differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. The medium provides the necessary components to support the chondrogenic differentiation process.
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Lab products found in correlation
Nh adipodiff medium, by Miltenyi Biotec (2 mentions)
Alcian blue, by Merck Group (2 mentions)
Heparin, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Alpha mem, by Merck Group (1 mentions)
β glycerol phosphate, by Merck Group (1 mentions)
L ascorbate 2 phosphate, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Stemmacs osteodiff media, by Miltenyi Biotec (1 mentions)
Stempro adipogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
15 ml polypropylene tube, by BD (1 mentions)
Nh osteodiff medium, by Miltenyi Biotec (1 mentions)
5 protocols using nh chondrodiff medium
1
Chondrogenic Differentiation of MSCs
2
Multilineage Differentiation Capacity
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To determine the adipogenic and osteogenic differentiation capacity, 4,000 cells/cm2 in passage 2 were seeded on a 12-well plate coated with fibronectin (2 μg/cm2; Sigma Aldrich) and allowed to grow confluent. For chondrogenic differentiation, 2.5 × 105 cells were seeded into a 15-ml tube and centrifuged (300g, 5 min) to form an aggregate. Then, in all conditions, the medium was changed to adipogenic, chondrogenic (NH AdipoDiff Medium or NH ChondroDiff Medium, both Miltenyi Biotech, Bergisch Gladbach, Germany, with 0.5% gentamycin), or osteogenic differentiation medium (MEM alpha, 2.5% HPL, 0.5% gentamycin, 1 U/ml heparin, 5 mM beta-glycerolphosphate, 0.1 μM dexamethasone, and 0.2 mM l -ascorbate-2-phosphate, all Sigma Aldrich), respectively. Cells were cultivated for 21 days, and the medium was changed every 2–3 days.
3
Multilineage Differentiation of UC-MSCs
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Multipotent differentiation was assessed evaluating the ability of UC-MSCs to differentiate into adipogenic, osteogenic and chondrogenic lineages under adapted media conditions. UC-MSCs were plated at 2 × 104cells/well in 24-multiwell culture plate and grown in StemPro® Adipogenesis Differentiation Kit (Gibco, ThermoFisher Scientific) for 3 weeks, replacing medium every 3 days. Adipogenesis was assessed using Oil Red O (Sigma-Aldrich) staining to detect intracellular lipid vacuoles. For osteogenic differentiation UC-MSCs were plated at 104 cells/well in 24-multiwell culture plate and grown in StemMACS OsteoDiff Media (Miltenyi Biotec GmbH, Germany) for 10 days, replacing medium every 3 days. Osteogenesis was assessed by alizarin red S (Sigma-Aldrich) staining to detect the deposition of intracellular calcium. Chondrogenic differentiation was assessed by plating 4 × 104 cells/well in NH ChondroDiff Medium (Miltenyi Biotec GmbH, Germany) for 3 weeks, replacing medium every 3 days. Chondrogenesis was confirmed using Alcian Blue staining (Sigma-Aldrich).
4
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.
5
Multilineage Differentiation of MSCs
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To induce differentiation of MSCs, specific medium was added to the cells according to the manufacturer’s instructions. Adipogenic differentiation was induced by NH Adipo Diff Medium (Miltenyi Biotec, Bergisch Gladbach). Osteogenic differentiation was achieved by NH OsteoDiff Medium (Miltenyi Biotec, Bergisch Gladbach), whereas chondrogenic differentiation was induced by NH ChondroDiff Medium (Miltenyi Biotec, Bergisch Gladbach). Each specific differentiation medium was changed every 2–3 days. Confirmation of differentiation of the cells to adipocytes, osteocytes and chondrocytes were performed by staining with Oil Red O staining solution, SIGMA FAST™ BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) tablets and Alcian blue-solution, respectively.
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