Stemxvivo osteogenic adipogenic base media

Manufactured by R&D Systems

StemXVivo Osteogenic/Adipogenic Base Media is a cell culture medium designed to support the growth and differentiation of mesenchymal stem cells (MSCs) into osteogenic (bone) and adipogenic (fat) lineages. The media provides the necessary nutrients and growth factors to maintain the undifferentiated state of MSCs and to promote their differentiation towards specific cell types.

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Lab products found in correlation

Stemxvivo adipogenic supplement, by R&D Systems (3 mentions) Stemxvivo human osteogenic supplement, by R&D Systems (3 mentions) Axio observer a1, by Zeiss (3 mentions) Adipogenic supplement, by R&D Systems (2 mentions) Cetylpyridinium chloride, by Merck Group (1 mentions) Alkaline phosphatase assay kit, by Abcam (1 mentions) Alizarin red staining solution, by Merck Group (1 mentions) Inverted microscope, by Nikon (1 mentions) Stemxvivo chondrogenic supplement, by R&D Systems (1 mentions) Crystal violet assay kit, by Abcam (1 mentions) Indomethacin, by R&D Systems (1 mentions) Oil red o solution, by Merck Group (1 mentions) Stemxvivo mouse rat osteogenic supplement 20, by R&D Systems (1 mentions) Its supplement, by R&D Systems (1 mentions) Osteogenic supplement, by R&D Systems (1 mentions)

Close spelling variants of this product

StemXVivo™ Human/Mouse Osteogenic/Adipogenic Base Media

12 protocols using stemxvivo osteogenic adipogenic base media

Adipogenic Differentiation of Mesenchymal and Epicardial Stem Cells

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Mesenchymal stem cells (MSCs) were isolated from adult (6–8 weeks) CFW femurs as described previously.¹⁶ Isolated cells were cultured in StemXvivo Osteogenic/Adipogenic Base media (R&D Systems) with Penicillin-Streptomycin (1:100). WT1⁺ EPDCs were isolated from heart explants of WT1^GFPCre/+ mice 2 days after MI. Cardiac cells (non-myocytes) were allowed to expand from heart explant cultures. After 2 weeks, GFP⁺ cells were isolated by FACS (FACS Aria III) and plated plated in fibronectin-coated (5 ng/ml for 2 hours at 37°C) wells of a 12 well plate (70,000 cells per well).
For enhancement of adipocyte differentiation in MSCs or EPDCs, culture medium (StemXvivo Osteogenic/Adipogenic Base media; R&D Systems) was supplemented with Adipogenic Supplement (R&D Systems, 1:20). ModRNAs were transfected every 3–4 days during adipogenic differentiation. For detection of oil droplets, cultures were stained with saturated Oil red O solution (Sigma).¹⁷ To quantitate oil red O staining, plates were dried and extracted with 1 ml 100% isopropanol. After 10 minutes incubation with gentle shaking, the OD₅₀₀ was recorded.

Zangi L., Oliveira M.S., Ye L.Y., Ma Q., Sultana N., Hadas Y., Chepurko E., Später D., Zhou B., Chew W.L., Ebina W., Abrial M., Wang Q.D., Pu W.T, & Chien K.R. (2016). An IGF1R-dependent pathway drives epicardial adipose tissue formation after myocardial injury. Circulation, 135(1), 59-72.

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Adipogenic Differentiation of Stem Cells

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To induce adipogenic differentiation, third-passage cells were treated with adipogenic medium for 9 days. Adipogenic medium consists of StemXVivo Osteogenic/Adipogenic Base Media (R&D Systems) supplemented with 10% of StemXVivo Adipogenic Supplement (R&D Systems) and 1% of P/S/A. To visualise formation of intracellular lipid droplets, cells were fixed with 4% PFA, incubated for 5 minutes with 60% isopropanol, and stained with 2% Oil Red O for 10 minutes. Excess stain was removed by washing with HBSS. Next, cells were counterstained with Mayer haematoxylin for 1 minute. After washing 3 times with distilled water, the positive cells containing lipid droplets were stained red.
Cell morphology was then assessed using an inverted epifluorescent microscope (Zeiss, Axio Observer A.1).

Marycz K., Kornicka K., Basinska K, & Czyrek A. (2015). Equine Metabolic Syndrome Affects Viability, Senescence, and Stress Factors of Equine Adipose-Derived Mesenchymal Stromal Stem Cells: New Insight into EqASCs Isolated from EMS Horses in the Context of Their Aging. Oxidative Medicine and Cellular Longevity, 2016, 4710326.

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Osteogenic Differentiation of MSCs

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The ability of culture-expanded MSCs to differentiate into the osteogenic lineage was validated according to earlier described methods (Herberg et al., 2013 ; Gregory et al., 2004 (link)). In brief, cells were plated in 12-well plates at 50000 cells/cm² and cultured in DMEM for 24 h. Culture medium was then aspirated and replaced with StemXVivo Osteogenic/Adipogenic Base Media (#CCM007 R&D Systems) supplemented with StemXVivo Human Osteogenic Supplement (#CCM008 R&D Systems). Treatment-containing medium was replaced 2 times per week. The early osteogenic differentiation marker, Alkaline Phosphatase, was assessed in cell culture media after 7 days using an Alkaline Phosphatase Assay Kit (#ab83369 Abcam). After 3 weeks, osteogenic differentiation was assessed by staining with Alizarin-Red Staining Solution; (#TMS-008-C Millipore Sigma). The cells were fixed with 10% formalin for 20 min at room temperature (RT) and stained with Alizarin-Red Staining Solution for 20 min at RT. Stained monolayers were visualized by phase-contrast microscopy using an inverted microscope (Nikon, Melville, NY). Differentiation was quantified as previously described (Ripoll and Bunnell, 2009 (link)). In brief, cells were destained using 10% cetylpyridinium chloride (#855561 Sigma-Aldrich) and collected samples analyzed using a microplate reader at 570 nm.

Elmansi A.M., Hussein K.A., Herrero S.M., Periyasamy-Thandavan S., Aguilar-Pérez A., Kondrikova G., Kondrikov D., Eisa N.H., Pierce J.L., Kaiser H., Ding K.H., Walker A.L., Jiang X., Bollag W.B., Elsalanty M., Zhong Q., Shi X.M., Su Y., Johnson M., Hunter M., Reitman C., Volkman B.F., Hamrick M.W., Isales C.M., Fulzele S., McGee-Lawrence M.E, & Hill W.D. (2020). Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells. Bone Reports, 12, 100270.

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Directed Adipose-Derived Stem Cell Differentiation

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Adipose derived hMSCs were maintained in low glucose DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C and 5% CO₂. Cells were maintained and expanded on plasma treated tissue culture dishes with media changes every three days. Passage 2–7 cells were used for all experiments. At 80–90% confluence, cells were detached with trypsin and plated at 30,000 cells/well on substrates in 24 well plates. For differentiation in mixed media, cells were incubated in StemXVivo Osteogenic/Adipogenic Base Media supplemented with human StemXVivo osteogenic supplement and StemXVivo adipogenic supplement (R&D systems) for 14 days. For differentiation in osteogenic media, cells were incubated in culture media supplemented with human StemXVivo osteogenic supplement for 14 days. For no induction control, cells were incubated in maintenance media.

Loye A.M., Kinser E.R., Bensouda S., Shayan M., Davis R., Wang R., Chen Z., Schwarz U.D., Schroers J, & Kyriakides T.R. (2018). Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass. Scientific Reports, 8, 8758.

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Chondrogenic Differentiation Protocol

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To induce chondrogenic differentiation, third-passage cells were cultured in chondrogenic media consisting of StemXVivo Osteogenic/Adipogenic Base Media (R&D Systems) supplemented with 10% of StemXVivo Chondrogenic Supplement and 1% of P/S/A for 11 days. After that, cells were fixed in 4% PFA, rinsed twice with distilled water for 5 min each, and stained at room temperature for 30 min with Safranin O solution rinsed 5 times with distilled water. We also evaluate quantitatively effectiveness of differentiation process by analysing expression profile of the following genes (RT-PCR): COMP (cartilage oligomeric matrix protein), COLL-2 (collagen type II), and VIM (vimentin). Sequences of primes are listed in Table 2.

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Determining KYN Effect on BMSC Density

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To determine whether KYN affected BMSCs density, we utilized a Crystal violet Assay Kit (#ab232855 Abcam) according to the manufacturer's protocol. In brief, BMSCs were plated in 96 well plates at 5000 cells/well and cultured in DMEM for 24 h. Culture medium was then aspirated and replaced with StemXVivo Osteogenic/Adipogenic Base Media (#CCM007 R&D Systems) supplemented with StemXVivo Human Osteogenic Supplement (#CCM008 R&D Systems) with or without different doses of KYN (10, 50, 200 μM). After 3 days, the culture media was removed and the cells were washed and stained with the Crystal Violet Staining solution for 20 min at RT. Then, the staining solution was removed and the remaining stain was solubilized for 20 min with the Solubilization Solution. Finally, the Crystal Violet stain was quantified using a microplate reader at 595 nm.

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Osteogenic and Adipogenic Differentiation of MSCs

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Osteogenesis was chemically induced by culture in StemXVivo Osteogenic/Adipogenic Base Media (R&D Systems), supplemented with StemXVivo Human Osteogenic Supplement (R&D Systems) and 1% P/S. Adipogenesis was chemically induced in high glucose (4.5 g/L) DMEM with 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 µg/mL insulin and 100 µM indomethacin (all reagents from Sigma), supplemented with 10% FBS and 1% P/S as described previously⁵³. MSCs were grown in osteogenic, adipogenic or control media for three weeks on collagen-I coated PA hydrogels for RT-qPCR assays, with seeding densities of 2000 and 1000 cells/cm², for primary and immortalised MSCs respectively (seeding rates were adjusted to account for a greater rate of proliferation in the immortalised cells). For cytochemistry assays, cells were seeded on tissue culture plastic (TCP) at densities of 1200 or 650 cells/cm², for primary and immortalised MSCs respectively.

Galarza Torre A., Shaw J.E., Wood A., Gilbert H.T., Dobre O., Genever P., Brennan K., Richardson S.M, & Swift J. (2018). An immortalised mesenchymal stem cell line maintains mechano-responsive behaviour and can be used as a reporter of substrate stiffness. Scientific Reports, 8, 8981.

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Adipogenic and Osteogenic Differentiation of MSCs

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WT and TNFR2 KO-MSCs were tested for their ability to differentiate into adipocytes and osteoblasts. Adipogenic differentiation was induced by culturing cells in a specific differentiation homemade medium for 21 days as already described [48 ]. Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Beldi G., Khosravi M., Abdelgawad M.E., Salomon B.L., Uzan G., Haouas H, & Naserian S. (2020). TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function. Stem Cell Research & Therapy, 11, 281.

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Adipogenic Differentiation of Cells

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When reaching 100% confluence, cells were treated as described above and then incubated for 3, 7, 14 or 21 days with adipogenic differentiation medium (StemXVivo^® Osteogenic/Adipogenic Base Media; R&D Systems) supplemented with the respective media supplement (StemXVivo^® Adipogenic Supplement; R&D Systems) to induce adipogenesis.

Ceccarelli S., Gerini G., Megiorni F., Pontecorvi P., Messina E., Camero S., Anastasiadou E., Romano E., Onesti M.G., Napoli C, & Marchese C. (2022). Inhibiting DNA methylation as a strategy to enhance adipose-derived stem cells differentiation: Focus on the role of Akt/mTOR and Wnt/β-catenin pathways on adipogenesis. Frontiers in Cell and Developmental Biology, 10, 926180.

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Osteogenic Differentiation of Human Cells

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DMEM medium was replaced by a StemXVivo Osteogenic/Adipogenic Base Media (#CCM007, R&D Systems) with 1% kanamycin sulfate and 5% StemXVivo human osteogenic supplement 20× (#CCM008, R&D Systems), which was replaced every 2–3 days. After 2 weeks, cells were washed with DPBS and fixed with 4% paraformaldehyde for immunological (osteocalcin) staining.

Dushime H., Moreno S.G., Linard C., Adrait A., Couté Y., Peltzer J., Messiaen S., Torres C., Bensemmane L., Lewandowski D., Romeo P.H., Petit V, & Gault N. (2023). Fetal Muse-based therapy prevents lethal radio-induced gastrointestinal syndrome by intestinal regeneration. Stem Cell Research & Therapy, 14, 201.

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