Adipogenic induction medium

Manufactured by Lonza

Sourced in United States

Adipogenic induction medium is a cell culture medium designed to promote the differentiation of stem cells or precursor cells into adipocytes (fat cells). It provides a defined set of components that support the conversion of these cells into mature fat cells.

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Lab products found in correlation

Osteogenic induction medium, by Lonza (3 mentions) Adipogenic maintenance medium, by Lonza (2 mentions) Oil red o, by Merck Group (2 mentions) Toluidine blue, by Fujifilm (2 mentions) Bone morphogenetic protein 6, by R&D Systems (2 mentions) Chondrogenic induction medium, by Lonza (2 mentions) Oil red o, by Muto Pure Chemicals (2 mentions) Osteogenic differentiation medium, by Lonza (1 mentions) Formalin, by Merck Group (1 mentions) Alizarin red, by Merck Group (1 mentions) Methanol, by Merck Group (1 mentions) Alizarin red staining, by Merck Group (1 mentions) Transforming growth factor β3, by Lonza (1 mentions) Oil red o staining, by Muto Pure Chemicals (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) Transforming growth factor β3, by R&D Systems (1 mentions) Alizarin red s staining, by Merck Group (1 mentions) Chondrogenic medium, by Lonza (1 mentions) Osteogenic medium, by Lonza (1 mentions) 6 well cell culture plate, by BD (1 mentions)

11 protocols using adipogenic induction medium

Evaluating Nanoparticle Effects on Stem Cell Differentiation

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hMSCs were seeded in 6-well tissue culture plates (cytochemical staining) or 12-well tissue culture plates (RNA isolation, both Greiner Bio one, Frickenhausen, Germany) at 2 × 10⁴ cells/cm² for adipogenic or 0.5 × 10⁴ cells/vm² for chondrogenic and osteogenic differentiation. Cells for adipogenic differentiation were cultivated in α–MEM until confluence and then differentiation was induced with Adipogenic Induction Medium (Lonza, Cologne, Germany). The medium was changed twice a week, altering between Adipogenic Induction Medium and adipogenic maintenance medium (Lonza, Cologne, Germany) for 3 weeks, followed by one week of cultivation in adipogenic maintenance medium. Osteogenic and chondrogenic differentiation was induced using NH Osteo Diff Medium/NH Chrondro Diff Medium (Miltenyi Biotec Bergisch Gladbach, Germany); the medium was changed twice a week. Analysis was performed after 24 days of cell culture.
Before starting the differentiation, all cells were incubated with 300 µg/mL nanoparticles for 24 h and non-treated samples served as a control. All experiments were also performed in α–MEM without differentiation factors (non-differentiated samples).

Brüstle I., Simmet T., Nienhaus G.U., Landfester K, & Mailänder V. (2015). Hematopoietic and mesenchymal stem cells: polymeric nanoparticle uptake and lineage differentiation. Beilstein Journal of Nanotechnology, 6, 383-395.

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Adipogenic and Osteogenic Potential of KNT Cells

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To confirm the adipogenic potential of young and elderly KNT cells, these cells were incubated in αMEM with 5% PLTMax until cells were confluent. Thereafter, KNT cells were cultured with adipogenic induction medium (Lonza, Basel, Switzerland). After 3 days, maintenance medium was added to cells, and three cycles of induction and maintenance media were completed. Cells were fixed with 10% formalin (Sigma-Aldrich) and stained with 0.5% Oil Red O (Sigma-Aldrich) in methanol (Sigma-Aldrich). To confirm the osteogenic potential of KNT cells, they were incubated in αMEM with 5% PLTMax until a confluent layer was achieved. Thereafter, osteogenic differentiation medium (Lonza) was added. Medium was changed every 3–4 days. After 17 days, cells were fixed in 10% formalin and stained with 10% alizarin red (Sigma-Aldrich).

Gladkova N., Umezu T., Imanishi S., Kawana C., Ohyashiki J.H, & Ohyashiki K. (2020). Effect of the extracellular component of bone marrow mesenchymal stromal cells from healthy donors on hematologic neoplasms and their angiogenesis. Human Cell, 33(3), 599-609.

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Adipogenic and Osteogenic Differentiation of hMSCs

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Example 3

Human mesenchymal stem cells (hMSCs) were cultured as instructed by the vendor. After cells were washed with PBS and trypsinized for 3-5 minutes, they were centrifuged in serum containing medium and followed with gentle resuspending in serum-free medium. The cells were then seeded onto transparent glass substrates and then incubated at 37° C. in a humidified atmosphere of 5% CO₂overnight. Adipogenic differentiation was induced by adipogenic induction medium and kept by induction/maintenance cycles as described in the Lonza protocol. Osteogenic differentiation was induced by osteogenic induction medium provided by Lonza.

US10017460B2. Compounds for promoting liposomal and cellular adhesion and compositions and methods of use thereof (2018-07-10). None [CA]. Inventors: Muhammad Naveed Yousaf [CA].

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Multilineage Differentiation of Cultured MSCs

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Cultured MSCs at three passages were harvested using Trypsin-EDTA (Gibco). For adipogenic differentiation, 1.0 × 10⁴ cells were transferred to a 24-well plate and cultured overnight in culture medium. Adherent cells were cultured in adipogenic induction medium (Lonza), which was changed every 3–4 days. After 14 days, oil red O staining (Muto Pure Chemicals) confirmed the differentiation of these cells into adipocytes. For osteogenic differentiation, 7.0 × 10³ cells were transferred to a 24-well plate and cultured overnight in culture medium. Adherent cells were cultured in osteogenic induction medium (Lonza), which was changed every 3–4 days. After 14 days, the differentiation of these cells into osteoblasts was assessed by alizarin red staining (Millipore). For chondrogenic differentiation, 5.0 × 10⁵ cells were transferred to a 15-mL tube and cultured in chondrogenic induction medium (Lonza) containing 10 ng/mL transforming growth factor-β3 (Lonza) and 500 ng/mL bone morphogenetic protein 6 (R&D Systems), which was changed every 3–4 days. After 21 days, chondrogenic differentiated cells were was analyzed by Toluidine blue (Wako) and Safranin O staining (TCI) staining.

Ogata Y., Mabuchi Y., Yoshida M., Suto E.G., Suzuki N., Muneta T., Sekiya I, & Akazawa C. (2015). Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration. PLoS ONE, 10(6), e0129096.

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Multilineage Differentiation of Mesenchymal Stem Cells

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For osteogenic and adipogenic-differentiation, adherent MSCs were cultured in osteogenic or adipogenic induction medium (Lonza), respectively, with twice weekly medium changes. After 21 days differentiation into osteoblasts was confirmed via alkaline phosphatase (ALP)-based enzymatic staining (Vector Laboratories, Burlingame, CA, United States) and Alizarin red S staining (Sigma-Aldrich, St. Louis, MO, United States). After 21 days differentiation into adipocytes was confirmed via Oil red O (Muto Pure Chemicals, Bunkyo, Tokyo, Japan) staining, respectively (Morikawa et al., 2009b (link); Houlihan et al., 2012 (link)).
To induce chondrogenic-differentiation, cultured cells or spheres were transferred into 15-mL tubes containing chondrogenic-induction medium (Lonza) with 10 ng/mL transforming growth factor-β3 (R&D Systems, Minneapolis, MN, United States) and 500 ng/mL bone morphogenetic protein-6 (R&D Systems). The medium was changed twice weekly. After 21 days, chondrogenic-differentiation was confirmed via staining with toluidine blue (Wako) (Morikawa et al., 2009b (link); Houlihan et al., 2012 (link)).

Niibe K., Ohori-Morita Y., Zhang M., Mabuchi Y., Matsuzaki Y, & Egusa H. (2020). A Shaking-Culture Method for Generating Bone Marrow Derived Mesenchymal Stromal/Stem Cell-Spheroids With Enhanced Multipotency in vitro. Frontiers in Bioengineering and Biotechnology, 8, 590332.

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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.

Kurose R., Sawai T., Oishi K., Liu X., Sasaki A., Kurose A., Kumagai N., Fujishima Y, & Ishibashi Y. (2017). Possibility of inhibiting arthritis and joint destruction by SSEA-3 positive cells derived from synovial tissue in rheumatoid arthritis. Regenerative Therapy, 7, 82-88.

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Quantifying Adipogenic Differentiation

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Cells were seeded at a density of 21,000/cm² and left to attach for 2–3 d until they reached confluency, upon which time they were stimulated with CSE and adipogenic induction medium (Lonza). The medium changes alternated between induction and maintenance medium following manufacturer’s instructions. After 21 d in culture, Oil Red O (Sigma-Aldrich, St Louis, MO, USA) was used to stain and quantify the resulting lipid droplets. Briefly, the cells were fixed with formalin, rinsed with PBS, and 60% isopropanol was used to eliminate background. The wells were allowed to dry and Oil Red O solution was added for 10 min. The wells were then rinsed with water and imaged. To elute the Oil Red O solution, 100% isopropanol was added for 10 min on an orbital shaker and the optical density of the solution was measured at 490 nm, 0.5 sec.

Wahl E.A., Schenck T.L., Machens H.G, & Egaña J.T. (2016). Acute stimulation of mesenchymal stem cells with cigarette smoke extract affects their migration, differentiation, and paracrine potential. Scientific Reports, 6, 22957.

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Culturing and Differentiating Mesenchymal Stem Cells

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Human mesenchymal stem cells (hMSCs) were cultured as instructed by the vendor. After cells were washed with PBS and trypsinized for 3–5 minutes, they were centrifuged in serum containing medium and followed with gentle resuspending in serum free medium. The cells were then seeded onto transparent glass substrates and then incubated at 37°C in a humidified atmosphere of 5% CO₂ overnight. Adipogenic differentiation was induced by adipogenic induction medium and kept by induction/maintenance cycles as described in the Lonza protocol. Osteogenic differentiation was induced by osteogenic induction medium provided by Lonza. 3T3 Swiss Abino Fibroblasts, RFP Expressing Human Neonatal Dermal Fibroblasts, and C3H/10T1/2 cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. NIH3T3/GFP cells were cultured in DMEM containing 10% FBS, 0.1 mM MEM Non-Essential Amino Acids, 2 mM L-glutamine, 10 μg/mL Blasticidin, and 1% penicillin/streptomycin. These cells were incubated at 37°C in a humidified atmosphere of 5% CO₂, and released from tissue culture plates using 0.05% trypsin in 0.53 mM EDTA.

Luo W., Pulsipher A., Dutta D., Lamb B.M, & Yousaf M.N. (2014). Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces. Scientific Reports, 4, 6313.

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Adipogenic Differentiation of hGMSCs

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CTR-hGMSCs and CBD-hGMSCs were seeded in a 6-well cell culture plate (Falcon) at a density of 2 7.5 × 10³ cells /cm² and incubated overnight. The medium was replaced with adipogenic induction medium (Lonza) composed by induction and maintenance medium. The medium After 28 days of treatment samples were fixed and stained with Oil Red O working solution (Sigma-Aldrich) and counterstained with hematoxylin to detect the oil globules. Images were collected using light microscopy Leica DMIL (Leica Microsystem, Milan, Italy) (Trubiani et al., 2016b (link)).

Libro R., Scionti D., Diomede F., Marchisio M., Grassi G., Pollastro F., Piattelli A., Bramanti P., Mazzon E, & Trubiani O. (2016). Cannabidiol Modulates the Immunophenotype and Inhibits the Activation of the Inflammasome in Human Gingival Mesenchymal Stem Cells. Frontiers in Physiology, 7, 559.

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Adipogenic Differentiation of ADSCs

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The collected ADSCs were seeded at 2.0 × 10 5 cells per well in 6-well plates (Nunc Cell-Culture Treated Multidishes; Thermo Fisher Scientific, Waltham, MA, USA) and cultured in cell-CM at 37 • C and 5% CO 2 until reaching 90% confluence. The cell-CM was then changed to adipogenic induction medium (Lonza, Walkersville, MD, USA), consisting of 4.5 g/L D-glucose, 100 µM indomethacin, 10 µg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 µM dexamethasone, and cultured for another 6 days. The cells were then formalin-fixed and stained with Oil red O (Muto Pure Chemicals, Co., Ltd., Tokyo, Japan) to assess the production of oil droplets. Cells cultured in cell-CM for 6 days were used as controls. The medium was changed every 3 days.

Fujimoto R., Murata D, & Nakayama K. (2022). Bio-3D printing of scaffold-free osteogenic and chondrogenic constructs using rat adipose-derived stromal cells. Frontiers in bioscience (Landmark edition), 27(2).

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