Mesenchymal stem cell osteogenic differentiation medium

Human immortalized mesenchymal stem cells (hiMSC) were gifted from Prof. Junya Toguchida (Kyoto University) [9 (link)]. Cells were maintained at 37 °C with 5 % CO₂ in high-glucose (4.5 g/L glucose) Dulbecco's modified Eagle's medium (DMEM; Nacalai tesque, Kyoto, Japan) supplemented with 10 % fetal bovine serum (FBS; Corning, Cellgro, New York, USA), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Nacalai tesque). To induce osteogenic differentiation, hiMSC cells were seeded in 6-well plates (3 × 10⁴ cells per well) with growth medium, and on the next day the growth medium was replaced by Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell, Heidelberg, Germany) with or without 100 nM vitamin D3 (VD3; Cayman Chemical, Michigan, USA). Osteogenic differentiation was continued for 1, 2, and 3 weeks.

DFATs at P2 were seeded on 24-well plates at a density of 6 × 10⁴ cells per well and cultured in the osteogenic differentiation induction medium (Mesenchymal Stem Cell Osteogenic Differentiation Medium: C-28013; Promo Cell). At 14 days of culture, samples were washed twice with PBS and fixed with 4% PFA for 60 min. After fixation, samples were washed twice with PBS and stained with 1% Alizarin red S (Sigma Aldrich) for 3 min. After being washed three times with distilled water, samples were photographed with the VB-7000 stereoscopic microscope (Keyence). The intensity of Alizarin red S staining was quantified by modifying the method of Gregory et al. [26 (link)]. Briefly, 10% acetic acid was added to each well and incubated at room temperature for 30 min with shaking. Loosely attached cells were collected with a cell scraper and transferred to a 1.5-mL tube. After heating at 85 °C for 10 min, samples were centrifuged at 20,000×g for 15 min and the supernatant was collected. Then, 50 μl of 5% ammonia water (pH 4.1–4.5) was added, and the absorbance at 405 nm was measured with the microplate reader (iMark).