iPSC-derived MSCs were cultured in MSC-GM (Lonza, Basel, Switzerland) media in 0.1% gelatin-coated tissue culture plates. For chondrogenic differentiation, cells were detached using 0.25% trypsin-EDTA (Invitrogen, CA, USA) and differentiated under 3D pellet culture conditions. Centrifuged MSCs were resuspended in 500 mL of chondrogenic differentiation media (100X Chondrogenic Supplement added to chondrogenic base media [all from R&D Systems]) in 15-mL tubes. Cells were centrifuged at 200 g for 5 min at room temperature, and the tubes were then placed upright in a 5% CO2-supplied 37 °C incubator with the lids slightly open to permit gas exchange. The medium was changed 3 times a week for 28 days. Differentiated chondrocytes were stained using Alcian blue solution.
Chondrogenic supplement
Manufactured by R&D Systems
Sourced in United States
The Chondrogenic supplement is a laboratory product designed to support the growth and development of chondrocytes, the cells responsible for the production of cartilage. This supplement provides the necessary nutrients and growth factors to facilitate the in vitro cultivation and differentiation of chondrocytes. The core function of this product is to create an optimal environment for the propagation and maintenance of chondrocyte cultures, which can be utilized in various research and development applications.
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Lab products found in correlation
Evos fl digital inverted fluorescence microscope, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Mscgm, by Lonza (1 mentions)
α mem, by R&D Systems (1 mentions)
Anti hfabp4, by R&D Systems (1 mentions)
Anti hoc, by R&D Systems (1 mentions)
Anti hacan, by R&D Systems (1 mentions)
Its supplement, by R&D Systems (1 mentions)
Stemxvivo human mouse chondrogenic supplement, by R&D Systems (1 mentions)
5 protocols using chondrogenic supplement
1
iPSC-derived MSCs Chondrogenic Differentiation
2
Multilineage Differentiation of hAMSCs
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The differentiation analysis of hAMSCs was done in both 2D and 3D cultures. Spheroids were dissociated into a single-cell suspension and seeded to the culture plates for cell expansion before differentiation assay. To evaluate osteogenic and adipogenic differentiation, cells were grown for 14 days in α-minimum essential medium (α-MEM) supplemented with both 10% FBS and osteogenic and adipogenic supplements, respectively (R&D Systems, USA). Chondrogenic differentiation was analyzed by growing cells in DMEM/F12 medium containing both insulin-transferrin-selenium (ITS) supplement (R&D Systems, USA) and chondrogenic supplement (R&D Systems, USA). A panel of antibodies consisting of anti-hFABP4, anti-hOC, and anti-hACAN was analyzed by immunofluorescence to define the mature phenotypes of adipocytes, osteocytes, and chondrocytes, respectively (R&D Systems, USA). Fluorescence for each antibody was revealed using EVOS™ FL Digital Inverted Fluorescence Microscope (Fisher Scientific, UK), and signal intensities were calculated with ImageJ software.
3
Multilineage Differentiation Assay of hAMSCs
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The differentiation assay was done in both 2D and 3D hAMSC cultures. The spheroid cells were dissociated into a single-cell suspension with 0.25% trypsin/EDTA and seeded to the culture plates for cell expansion. Osteogenic differentiation and adipogenic differentiation were evaluated by growing the cells for 14 days in α MEM with 10% FBS supplemented with osteogenic and adipogenic supplements, respectively (R&D Systems, USA). Medium with DMEM/F12 containing both ITS supplement (R&D Systems, USA) and chondrogenic supplement (R&D Systems, USA) was used for chondrogenic differentiation. A panel of antibodies consisting of anti-mFABP4, anti-hACAN, and anti-hOC was assayed by immunofluorescence to define the mature phenotypes of adipocytes, chondrocytes, and osteocytes, respectively (R&D Systems, USA). Samples were analyzed using an EVOS™ FL Digital Inverted Fluorescence Microscope (Fisher Scientific, Paisley, Scotland, UK). Signal intensities were calculated with ImageJ software.
4
Chondrogenic Differentiation of MSCs
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MSCs were transferred in a 15 ml conical tubes (250,000 cells per tube), centrifuged at 200g for 5 min and resuspended in D-MEM/F12. MSCs were centrifuged again and resupended in 0.5 ml of D-MEM/F12 containing 100 mg ml−1 penicillin/streptomycin, 1% ITS supplement, 1 × chondrogenic supplement (R&D Systems), with or without murine IL-1β (1 ng ml−1). Cells were centrifuged on more time at 200g for 5 min to form a pellet. The cap of the tubes were loosen to allow gas exchange and the tubes were incubated upright at 37 °C with 5% CO2. The medium was replaced every 3 days (without IL-1β). After 28 days, the spheroids were washed with PBS and fixed with 10% formalin for 60 min. Spheroids were washed twice with water and stained with Alican Blue 8 GX (Sigma-Aldrich, 0.1 mg ml−1 in ethanol/acetic acid solution (3:2)) overnight at room temperature in the dark. Spheroids were destained three times with an ethanol/acetic acid solution (3:2) and resuspended in PBS, before imaging.
5
Chondrogenic Differentiation of DPSCs and BM-MSCs
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Chondrogenic differentiation of DPSCs and BM-MSCs was induced according to the manufacturer’s instructions (StemXVivo Human/Mouse chondrogenic supplement, R&D systems, BioTechne, Minneapolis, MN, USA). A pellet containing 2.5 × 105 cells in a 15 mL conical tube was subjected to chondrogenic differentiation medium consisting of DMEM/F12 supplemented with 1% insulin transferrin selenite (R&D systems) and 1% chondrogenic supplement (R&D systems). This supplement consists of dexamethasone, ascorbate-phosphate, proline, pyruvate and TGF-β3 with concentrations determined and validated by the manufacturer. To determine the effect of L-PRF on the chondrogenic differentiation, L-PRF ex (3%) and L-PRF CM (5% and 25%) were added to the differentiation medium. Positive and negative controls contained standard differentiation medium with or without the chondrogenic supplement respectively. Every 2–3 days, the medium was changed. The cells were allowed to differentiate for 21 days, after which the pellets were either fixed with 4% PFA for immunohistochemical (IHC) analysis or with 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.3) at 4 °C for TEM processing. Percentage alcian blue and aggrecan stained area was quantified using Image J (The National Institute of Health, MD, USA).
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