cMSCs and kPSCs were cultured in adipogenic, osteogenic and chondrogenic medium according to manufacturer’s protocol (Lonza). Calcium deposits were shown with Alizarin Red in the osteogenic differentiation assay. In the adipogenic differentiation assay, lipid droplets were stained using Oil Red O. In the chondrogenic differentiation assay, cell pellets were formalin-fixed (4% PFA o/n) and embedded in paraffin. 5 μm sections were de-parafinnized, rehydrated and incubated with 1% toluidine blue for 20 minutes. All differentiations were analysed with an inverted bright-field microscope (Leica DFC 295).
Chondrogenic medium
Manufactured by Lonza
Sourced in Switzerland, United States
Chondrogenic medium is a specialized cell culture medium designed to support the growth and differentiation of chondrocytes, which are the primary cells found in cartilage tissue. This medium provides the necessary nutrients and growth factors to promote the development and maintenance of cartilage-like structures in in vitro cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Osteogenic medium, by Lonza (2 mentions)
Dfc295, by Leica (1 mentions)
Adipocyte medium, by ZenBio (1 mentions)
Mesencult medium, by STEMCELL (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Dexamethasone (dex), by STEMCELL (1 mentions)
β glycerophosphate, by STEMCELL (1 mentions)
Mesencult msc basal medium, by STEMCELL (1 mentions)
Ascorbic acid, by STEMCELL (1 mentions)
Tgf β3, by R&D Systems (1 mentions)
Adipogenic induction medium, by Lonza (1 mentions)
Adipogenic maintenance medium, by Lonza (1 mentions)
Calcein solution, by Merck Group (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate bcip, by Merck Group (1 mentions)
Isobutylmethylxanthine ibmx, by Merck Group (1 mentions)
Dmem low glucose, by Merck Group (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
10 protocols using chondrogenic medium
1
Multipotent Stem Cell Differentiation Assays
2
Multilineage Differentiation Assay
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To evaluate adipogenic differentiation, cells were cultured in differentiation medium (Zen-Bio, NC, USA). After 7 days, 50% of the medium was replaced with the adipocyte medium (Zen-Bio) and this was repeated every 3 days. After 3 weeks of incubation, the adipogenic differentiation was confirmed by microscopic observation of intracellular lipid droplets with the aid of the Oil Red O staining, and by qPCR analysis. Osteogenic differentiation was induced by culturing the cells in osteocyte differentiation medium (Zen-Bio). Differentiation was examined using Alizarin Red staining, and qPCR analysis. To evaluate chondrogenic differentiation, 2 × 105 cells were centrifuged at 400 × g for 10 min. The resulting pellets were cultured in chondrogenic medium (Lonza, Basel, Switzerland) for 14 days. Then the pellets were harvested and analyzed by qPCR analysis.
3
Isolation and Characterization of Bone Marrow Stromal Cells
4
Multilineage Differentiation of ASCs
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The potential of ASC to differentiate into osteoblasts and adipocytes was confirmed in monolayer culture. To induce adipogenic differentiation, cells were cultured in MesenCult™ adipogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) supplemented with MesenCult® MSC basal medium (STEMCELL Technologies, Canada). To induce osteogenic differentiation, we used MesenCult™ osteogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) in the osteogenic medium containing 10−4 M dexamethasone (STEMCELL Technologies, Canada), 1 M β-glycerophosphate (STEMCELL Technologies, Canada), and 10 mg/ml ascorbic acid (STEMCELL Technologies, Canada). Media from both cultures were replaced every 3 d for 21 d in total. The differentiation potential for adipogenesis and the formation of intracellular lipid droplets were assessed by Oil Red O staining after fixation in 10% formalin. The differentiation potential for osteogenesis was assessed by Alizarin Red S (ARS) staining after fixation in 10% formalin. To induce chondrogenic differentiation, we used a chondrogenic medium (LONZA, Switzerland). For histological analysis, pellets were embedded in paraffin and sectioned. Chondrogenic differentiation was assessed by Masson trichrome staining. Morphology and differentiation potential of ASCs are represented for one individual donor from three independent donors.
5
Chondrogenic Pellet Culture with Rapamycin
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Pellet culture was performed as described previously.76 (link) Pellets were made in 0.5 mL of chondrogenic medium (Lonza, Allendale, NJ, USA) supplemented with 10 ng/mL transforming growth factor-β3 (TGF-β3; R&D System, Minneapolis, MN, USA), with or without 10 nM rapamycin. The pellets were incubated at 37°C in 5% CO2 and the medium was changed every 3 days. Pellets were harvested after 14 days in culture. Cultured micromass pellets were fixed with 10% neutral-buffered formalin containing 0.1% cetylpyridinium chloride. The longest and shortest diameters of the pellets were measured using ImageJ software (NIH). Average diameters of the pellets were obtained by dividing the sum of diameters measured for each pellet by the number of measurements.77 (link), 78 (link) Pellets were stained with 1% Alcian Blue (pH 1.0) for 30 min in order to stain the highly sulfated proteoglycans that are characteristic of cartilaginous matrices. Data from three replicate plates for four independent experiments were analyzed. Total RNA was extracted for qRT-PCR analysis. The expression patterns of chondrogenic genes expressing collagen type 2A1 (Col2A1) and aggrecan were analyzed.
6
Chondrocyte Culture under Temperature Stress
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For monolayer culture, the expanded chondrocytes were trypsinized and sub-cultured at 1 × 104 cells/cm2 in culture medium into 24 culture dishes (35 mm diameter). For pellet cultures, chondrocytes were trypsinized, washed with culture medium, and resuspended in chondrogenic medium (Lonza, Walkersville, MD, USA: chondrogenic basal medium, plus ITS + supplement, ascorbate, dexamethasone, l -glutamine, sodium pyruvate, proline, and GA-1000) supplemented with 10 ng/mL of recombinant human transforming growth factor-beta 3 (R&D Systems Inc., Minneapolis, MN, USA). Aliquots of 2.5 × 105 cells in 500 μL of the chondrogenic medium in 24 15-mL-polypropylene conical tubes were centrifuged at 250 × g for 5 min to form a cell pellet. The monolayer culture cells and the pelleted cells were pre-cultured at 37°C for 3 days. After pre-culture, the dishes and the tubes were exposed to three distinct temperatures for 3 additional days by transferring them into three distinct CO2 incubators set at 32°C, 37°C, and 41°C (8 dishes and tubes for each group). These culture temperatures were defined as follows: 32°C, the physiological intra-articular temperature [29 (link),30 (link)]; 37°C, the inner body temperature, which is conventionally used; and 41°C, high temperature, the threshold temperature for mammalian cell survival [34 (link),35 (link)].
7
Chondrogenic Differentiation via 3D Pellet Culture
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To induce chondrogenic differentiation, three-dimensional pellet culture was performed. In a 15 ml tube, 3 × 105 cells were pelleted by centrifugation. Unsuspended cell pellets were cultured for 19 days in chondrogenic medium (Lonza) composed of basic medium supplemented with dexamethasone, ascorbate, ITS + supplement, pyruvate, proline, GA-1000, L-glutamine and recombinant human transforming growth factor-β3. For histological analysis, pellets were immersed in paraffin, sectioned and stained with Masson trichrome method.
8
Multi-lineage Differentiation of SSEA-3+ Cells
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To investigate the multipotency of SSEA-3 positive cells in in vitro, osteogenic differentiation was performed using osteogenic medium (Lonza, Walkersville, MD, USA) consisting of dexamethasone, ascorbic acid, and β-glycerophosphate in a 6-well dish. In vitro adipogenic differentiation was also performed using adipogenic induction medium (Lonza) consisting of insulin, dexamethasone, indomethacin, and IBMX (3-isobutyl-methyl-xanthine) and adipogenic maintenance medium (Lonza) consisting of insulin in a 6-well dish. For in vitro chondrogenic differentiation, we utilized high-density three-dimensional micromass culture [21] (link), [22] (link), in which cells were trypsinized and resuspended at a density of 1 × 105 cells/10 μl. Ten microliter droplets were seeded in culture dishes and allowed to form cell aggregates and substratum at 37 °C for two and a half hours. Chondrogenic medium (Lonza), consisting of ITS + premix (6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 μg/mL selenous acid, 5.33 μg/mL linoleic acid, and 1.25 mg/mL bovine serum albumin), pyruvate (1 mmol/L), ascorbate 2-phosphate (0.17 mmol/L), proline (0.35 mmol/L), dexamethasone (0.1 μmol/L) and recombinant human TGF-β3 (10 ng/mL) was then carefully added around the cell aggregates. This Chondrogenic medium was replenished every three days.
9
Chondrogenic Differentiation of MSCs
10
Mesenchymal Differentiation Capacity Evaluation
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To confirm the mesenchymal activity of those cells that grew out naturally from the lipoaspirate clusters, while under culture conditions, their in vitro differentiation capacity was studied according to Noël et al. (26) . Briefly, 10 × 10 3 cells/ cm 2 were cultured in DMEM-low glucose supplemented with 10% FBS, 0.5 mM isobutyl-methyl xanthine (IBMX; Sigma-Aldrich), 200 μM indomethacin, 1 μM dexamethasone, and 10 μg/ml insulin (all from Sigma-Aldrich); after 2 weeks they were stained with fresh Oil red O solution (Sigma-Aldrich) for adipogenesis. After 3 weeks of culture in DMEM-low glucose supplemented with 10% FBS, 10 mM b-glycerophosphate, 0.2 mM ascorbic acid, and 10 nM dexamethasone (all from Sigma-Aldrich), for osteogenic differentiation, cells were stained with calcein solution (Sigma-Aldrich) to evidence mineralization (16) and with 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma-Aldrich) to evidence alkaline phosphatase activity (8). To demonstrate chondrogenic differentiation, pellets of 5 × 10 5 cells were cultured for 3 weeks in chondrogenic medium (Lonza, Cologne, Germany), formalin-fixed, embedded in paraffin, and immunostained for type II collagen by incubation with rabbit polyclonal anti-collagen type II primary antibody 1 mg/ml (Biogenesis, Oxford, UK) and with secondary antibody conjugated to peroxidase (Vector Laboratories).
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