Nh adipodiff medium

To determine the adipogenic and osteogenic differentiation capacity, 4,000 cells/cm² in passage 2 were seeded on a 12-well plate coated with fibronectin (2 μg/cm²; Sigma Aldrich) and allowed to grow confluent. For chondrogenic differentiation, 2.5 × 10⁵ cells were seeded into a 15-ml tube and centrifuged (300g, 5 min) to form an aggregate. Then, in all conditions, the medium was changed to adipogenic, chondrogenic (NH AdipoDiff Medium or NH ChondroDiff Medium, both Miltenyi Biotech, Bergisch Gladbach, Germany, with 0.5% gentamycin), or osteogenic differentiation medium (MEM alpha, 2.5% HPL, 0.5% gentamycin, 1 U/ml heparin, 5 mM beta-glycerolphosphate, 0.1 μM dexamethasone, and 0.2 mM l-ascorbate-2-phosphate, all Sigma Aldrich), respectively. Cells were cultivated for 21 days, and the medium was changed every 2–3 days.

hASCs differentiation capability was assessed by investigating osteogenic and adipogenic induction. For osteogenic differentiation, cells were seeded into six-well culture plates and at a density of 5.0 × 10³ cells/cm² and cultivated in NH OsteoDiff medium (Miltenyi Biotec, GmBH) for two weeks. Osteogenic potential was assessed by alkaline phosphatase activity with SIGMA FAST BCIP/NBT substrate (SIGMA-Aldrich, USA).
For adipogenic differentiation, cells were placed into six-well culture plates at a density of 5.0 × 10³ cells/cm² and cultivated in NH AdipoDiff medium (Miltenyi Biotec, GmBH) for three weeks. Adipogenic potential was assessed by Oil Red O (SIGMA-Aldrich, USA) staining.

To induce differentiation of MSCs, specific medium was added to the cells according to the manufacturer’s instructions. Adipogenic differentiation was induced by NH Adipo Diff Medium (Miltenyi Biotec, Bergisch Gladbach). Osteogenic differentiation was achieved by NH OsteoDiff Medium (Miltenyi Biotec, Bergisch Gladbach), whereas chondrogenic differentiation was induced by NH ChondroDiff Medium (Miltenyi Biotec, Bergisch Gladbach). Each specific differentiation medium was changed every 2–3 days. Confirmation of differentiation of the cells to adipocytes, osteocytes and chondrocytes were performed by staining with Oil Red O staining solution, SIGMA FAST™ BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) tablets and Alcian blue-solution, respectively.

Adipogenic differentiation assay was performed as previously described [39 (link)]. The paired culture-expanded IC-BM and VB-BM MSCs at passage 3 were equally distributed in triplicates with a density of 4x10⁴ per well of 48-wells/plate and cultured in NH AdipoDiff medium (Miltenyi Biotec). Following 21 days of culture, one well was used for the staining of cells with Oil Red O and two wells for the staining of cells with Nile Red/DAPI. The images were captured using an inverted light microscope (IX71 Olympus, Southend-on-Sea, UK) in combination with a fluorescent generator (for Nile Red/DAPI) and an Olympus Digital camera.

To further characterize the differentiation potential of isolated CB-MSCs, cells at passage of 5 were selected for following treatments as previously described (Ma et al., 2019 (link)). Briefly, cells were allowed to grow to ~100% confluency in tissue culture plates before replacing regular culture medium with NH OsteoDiff medium (Miltenyi Biotec) and NH AdipoDiff medium (Miltenyi Biotec) to induce the differentiation into adipocytes and osteoblasts, respectively. To induce chondrogenic differentiation, ~2 × 10⁶ cells were harvested and cultured in NH ChodroDiff medium (Miltenyi Biotec) in 15 mL Falcon tubes. All induction experiments were performed according to the manufacturer's instructions with each medium replaced twice a week. After in vitro induction, cells in tissue culture plates were stained using Alizarin Red (Sigma-Aldrich) and Oil Red O (Sigma-Aldrich) to examine osteogenic and adipogenic differentiation, respectively. For cells cultured in 15 mL Falcon tubes, cells were processed as previously reported in our lab (Ma et al., 2019 (link)), and finally stained with Alcian Blue (Sigma-Aldrich) to examine chondrogenic differentiation. All samples were imaged using phase contrast microphotography.