Osteogenic induction medium

Manufactured by Lonza

Sourced in United States

Osteogenic induction medium is a specialized cell culture medium designed to support the differentiation of cells towards the osteogenic lineage. It contains a combination of growth factors and other components that promote the development of bone cells from progenitor or stem cells.

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Lab products found in correlation

Alizarin red s, by Merck Group (3 mentions) Adipogenic induction medium, by Lonza (3 mentions) Alizarin red staining, by Merck Group (2 mentions) Chondrogenic induction medium, by Lonza (2 mentions) Oil red o staining, by Muto Pure Chemicals (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Complete maintenance medium, by Lonza (2 mentions) Induction medium, by Lonza (2 mentions) Maintenance medium, by Lonza (2 mentions) Osteogenic singlequots kit, by Lonza (1 mentions) Hmscs, by Lonza (1 mentions) Mscgm, by Lonza (1 mentions) P nitrophenyl phosphate, by Merck Group (1 mentions) Protein assay, by Bio-Rad (1 mentions) Tgf β3, by Lonza (1 mentions) Toluidine blue, by Fujifilm (1 mentions) Bone morphogenetic protein 6, by R&D Systems (1 mentions) Transforming growth factor β3, by Lonza (1 mentions) D7000, by Nikon (1 mentions) Collagen 1, by Corning (1 mentions)

14 protocols using osteogenic induction medium

Alizarin Red S Staining for Osteogenic Differentiation

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Cells were seeded at a density of 3,100/cm² and left to attach overnight before being stimulated with CSE and osteogenic induction medium (Lonza). After 21 d in culture, cells were stained with Alizarin red S (Sigma-Aldrich) to visualize and quantify calcium deposition. Briefly, the cells were fixed with formalin, rinsed with deionized water, and stained with 0.5% Alizarin red S solution. The cells were rinsed with deionized water and imaged. The quantification of the staining was carried out following the method of44 (link) and the absorbance was read at 530 nm.

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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Osteogenic Differentiation of PαS Cells

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Osteogenic differentiation of the PαS cells was performed using Osteogenic Induction Medium (Lonza) and supplements from the Osteogenic SingleQuots Kit (Lonza), according to the manufacturer's protocol. Briefly, PαS cells were seeded at 30,000/well in maintenance medium and allowed to attach. Next, the medium was changed to the Osteogenic Induction Medium in the presence or absence of OSM. Alizarin Red S staining was used to evaluate osteoblastic differentiation by detecting mineralized nodules. Alizarin Red S (Sigma) was dissolved in distilled water, and the pH was adjusted to pH 6.38 by the addition of 0.25% ammonium hydroxide. Cultured cells were washed with PBS and fixed in 95% ethanol for 10 min at room temperature. After washing with distilled water, the cells were incubated in Alizarin Red S solution for 10 min at room temperature. The cells were then gently washed with distilled water, 95% and 100% ethanol.

Sato F., Miyaoka Y., Miyajima A, & Tanaka M. (2014). Oncostatin M Maintains the Hematopoietic Microenvironment in the Bone Marrow by Modulating Adipogenesis and Osteogenesis. PLoS ONE, 9(12), e116209.

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Osteogenic Differentiation of hMSCs

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The hMSCs were purchased from Lonza (Walkersville, MD). After thawing, they were added to pre-warmed mesenchymal stem cell growth medium (MSCGM™), including mesenchymal stem cell basal medium supplemented with l-glutamine, gentamicin, and amphotericin provided by Lonza and incubated at 37 °C in a 5% CO₂ atmosphere. Upon 90 % confluency, trypsin/EDTA solution was used to detach cells for seeding.
Prior to seeding, the disks were autoclaved at 121 °C for 1 h. The samples were placed in 24-well plates, MSCGM™ was added to wet the samples, and the plates were incubated for 45 min. The cells were aspirated in media and seeded directly onto the samples (12,000 cells/sample), then incubated for 15 min to facilitate cell attachment. MSCGM™ was then added at 1 mL per well, and cultures were kept at 37 °C for the rest of the study. On day 3, the MSCGM™ was substituted with osteogenic induction medium, purchased from Lonza as a kit. The medium kit provided hMSC differentiation basal medium, dexamethasone, L-glutamine, ascorbate, β-glycerophosphate, Mesenchymal Cell Growth Supplement (MCGS™), and penicillin/streptomycin. This differentiation media was changed every 2-3 days.

Vahabzadeh S., Robertson S, & Bose S. (2021). Beta-phase Stabilization and Increased Osteogenic Differentiation of Stem Cells by Solid-State Synthesized Magnesium Tricalcium Phosphate. Journal of materials research, 36(15), 3041-3049.

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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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Osteogenic Differentiation of MSCs

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MSCs seeded in 2D or 3D substrates at a density of 8 × 10⁴ cells/well were cultured for 3 days in growth medium or 15 days in osteogenic induction medium (Lonza) consisting of MSC basal medium and the Single Quots^© osteogenic supplements containing FBS, l-glutamine, penicillin/streptomycin, dexamethasone, ascorbate and β-glycerophosphate. To prevent nutrient exhaustion, osteogenic induction medium was partially replaced every 3 days with an equal volume of fresh medium. Alkaline phosphatase (ALP) activity was assayed in cell layers by determining the release of p-nitrophenol from p-nitrophenylphosphate (Sigma) at 37 °C and a pH of 10.5. The data were normalized to the total protein amounts in cell layers determined by the Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA), based on the Bradford dye-binding method, using BSA as standard.

Vallés G., Bensiamar F., Crespo L., Arruebo M., Vilaboa N, & Saldaña L. (2015). Topographical cues regulate the crosstalk between MSCs and macrophages. Biomaterials, 37, 124-133.

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Differentiation of Mesenchymal Stem Cells

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Cultured ACL- and BM-MSCs at passage 3 were harvested using trypsin–EDTA (Gibco), transferred to a 24-well plate and grown overnight in culture medium. The cell numbers used were as follows: 2.0 × 10⁴ cells (for adipogenesis) and 1.5 × 10⁴ cells (for osteogenesis). For adipogenic differentiation, adherent cells were cultured in adipogenic induction and maintenance medium (Lonza), which was changed every 3–4 days. After 14 days, Oil red O staining (Muto Pure Chemicals) was used to confirm the differentiation of the cells into adipocytes. For osteogenic differentiation, adherent cells were cultured in osteogenic induction medium (Lonza), which was changed every 3–4 days. After 14 days, the differentiation of the cells into osteoblasts was assessed by Alizarin Red staining (Millipore). For chondrogenic differentiation, 2.0 × 10⁵ cells were transferred to a 1.5 mL tube and cultured in chondrogenic induction medium (Lonza) containing 10 ng/mL TGF-β3 (Lonza) and 500 ng/mL BMP-6 (R&D Systems), which was changed every 3–4 days.

Ogata Y., Mabuchi Y., Shinoda K., Horiike Y., Mizuno M., Otabe K., Suto E.G., Suzuki N., Sekiya I, & Akazawa C. (2018). Anterior cruciate ligament-derived mesenchymal stromal cells have a propensity to differentiate into the ligament lineage. Regenerative Therapy, 8, 20-28.

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Osteogenic and Adipogenic Differentiation of MSCs

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As a control of MSC characteristics, osteogenic differentiation was induced by seeding MSCs at a density of 3100 cells/cm² and by culturing them for 28 days in an osteogenic induction medium (Lonza, USA). After 28 days, the samples were fixed in 4% paraformaldehyde and then embedded in paraffin before being stained with alizarin red. To induce adipocyte differentiation, 21,000 MSCs/cm² were seeded onto a 24-well plate. When 100% confluence was reached, 3 cycles of induction/maintenance were performed. One cycle of induction/maintenance consisted in 3 day-culture in induction medium (Lonza, USA), followed by 1 to 3 days of culture in maintenance medium (Lonza). After 3 cycles of induction/maintenance, the cells were cultured for 7 days in complete maintenance medium (Lonza, USA) before being stained with oil red.

Laroye C., Boufenzer A., Jolly L., Cunat L., Alauzet C., Merlin J.L., Yguel C., Bensoussan D., Reppel L, & Gibot S. (2019). Bone marrow vs Wharton’s jelly mesenchymal stem cells in experimental sepsis: a comparative study. Stem Cell Research & Therapy, 10, 192.

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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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Osteogenic Differentiation of Stem Cells

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Osteogenic differentiation (OsD) of stem cells was performed at defined passages 3–5. To promote osteogenic differentiation, the cells were seeded at a density of 3.1 × 10³ cells per cm² into 75 T flask and cultured in ADSCGM for ADSC, MSCGM for hMSC and DMEM for TMSC until they reached 70–80% confluence. As soon as subconfluence was reached, osteogenic differentiation of the cells was induced by feeding them for 2 weeks, twice a week with osteogenic induction medium (Lonza, Basel, Switzerland) for ADSC and hMSC. DMEM with 50 µg/ml ascorbic acid, 10 mM B-glycerophosphate, 10 nM dexamethasone was used for TMSC osteogenic differentiation^{12 (link)}.

Kim M.S., Lee M.H., Kwon B.J., Kim D., Koo M.A., Seon G.M, & Park J.C. (2017). Homogeneity evaluation of mesenchymal stem cells based on electrotaxis analysis. Scientific Reports, 7, 9582.

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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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