Adipogenesis differentiation kit

When cells reached the proper confluence, the medium was replaced with differentiation medium from Adipogenesis Differentiation Kit (Gibco). After 14 days of differentiation, cells were fixed with 4% PFA for 30 minutes and washed with PBS and then 60% isopropanol was added for 5 minutes.
Staining was made of 99% isopropanol and Oil Red O (Sigma-Aldrich). The resulting solution was diluted in distilled water (3 : 2). Isopropanol was removed and the staining was added after 10 minutes of incubation on the cells for 5 minutes to verify positive effect of differentiation.

When cells reached the proper confluence, the medium was replaced with differentiation medium from the Adipogenesis Differentiation Kit (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).). After 14 days of differentiation, cells were fixed with 4% PFA for 30 min and washed with PBS, and then 60% isopropanol was added for 5 min.
Staining was achieved with 99% isopropanol plus Oil Red O (Sigma-Aldrich, Saint Louis, MO, USA). The resulting solution was diluted in distilled water (3:2). Isopropanol was removed and the stain added after 10 min of incubation of the cells for 5 min to verify the positive effect of differentiation.

After expansion in the mini-bioreactor, cells were evaluated regarding their potential to differentiate into adipocytes, osteocytes and chondrocytes. For adipogenic and osteogenic differentiation, cells were plated in duplicate at a density of 3000 cells/cm² on a 24-well plate (pre-coated with CELLstart, dilution 1:100) with culture medium. At confluence, cells were induced to differentiate into osteocytes and adipocytes using StemPro^TM Osteogenesis and Adipogenesis Differentiation Kit (Gibco, Grand Island, NY, United States), respectively. Culture medium was exchanged twice a week. Adipogenesis was observed after 14 days for lipid droplets (Sudan II-Scarlet) and osteogenesis after 21 days for mineralized bone matrix deposition (von Kossa). For chondrogenic differentiation, 1 × 10⁷ cells were plated in droplets (5 μL) on Ultra-Low attachment multi- well plates (Corning, Lowell, United States). The plate was left in the incubator for 30 min and afterward StemPro^TM Chondrogenesis Differentiation medium (Gibco, Grand Island, NY, United States) was added. Total medium exchange was performed two times a week for 15 days. Cells were then stained with Alcian blue (1%, Sigma- Aldrich, St Louis, MO, United States) for 2 h to assess proteoglycan synthesis (Mizukami et al., 2016 (link)).

Balb/c mice (2–3 weeks old) were decapitated, and the femur and tibia were carefully removed. Bone marrow cells were flushed with a syringe (27‐gauge) inserted into one end of the bone and cultured at a density of 3 × 10⁵ cell/cm² with low‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 15% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). After 2 hours, nonadherent cells were removed via medium changes, which were repeated every 3 days until the cultured cells reached 70%–80% confluence. Cells were then digested with TrypLE Express (Gibco) for 2 minutes and passaged. All experiments used MSCs at passage 5–8.
To evaluate their multipotent differentiation potential, MSCs were incubated at passage 5 in a 12‐well plate at a density of 5 × 10³ cells/cm² with osteogenesis differentiation medium (Gibco) or at a density of 1 × 10⁴ cells/cm² with an adipogenesis differentiation kit (Gibco) for 3 weeks. According to the manufacturer's instructions, other MSCs were seeded at a higher density of 1 × 10⁷ cells per milliliter to formulate a micromass and cultured with a STEMPRO chondrogenesis differentiation kit (Gibco) with medium changes every 3 days for 2 weeks. Osteoblasts were stained using Alizarin Red S (Sigma), adipocytes were stained with Oil Red O (Sigma), and chondrogenesis pellets were stained with Toluidine Blue (Sigma).

At passage three, human BM-derived MSCs were cultured with the Adipogenesis Differentiation Kit (Gibco, USA) according to the manufacturer’s instructions. At day 14 of differentiation, oil red O staining was carried out. Briefly, cells were washed with PBS and fixed in 4% paraformaldehyde for 30 minutes. After washing twice with PBS, cells were stained with 0.3% oil red O solution (Sigma Aldrich, USA) for 20 minutes at room temperature. Staining was quantified by extracting oil red O from the stained cells with isopropanol, followed by determining the optical density (OD) values of the solution at 518 nm. The NAC treatment method was performed according to the method published by Tormos et al [13 (link)]. The NAC treatment was started on Day 2 of differentiation and lasted until Day 14. On Day 1, the differentiation and NAC+differentiation groups were treated with the Adipogenesis Differentiation Kit, while the undifferentiated and NAC-treated groups were not subjected to adipocyte differentiation. On Day 2, cells were exposed for 4 hours prior to ROS measurement to NAC treatment (5 mM) or to PBS for control group.

Adipogenic and osteogenic differentiation assays at the fourth passage were conducted to detect the multilineage differentiation potential of the isolated human UC‐MSCs. UC‐MSCs were induced to differentiate using an Adipogenesis Differentiation Kit (A1007001, Gibco) and Osteogenesis Differentiation Kit (A1007201, Gibco). Lipid accumulation was detected at 7–14 days after induction culture by Oil Red O staining. For osteogenic differentiation, the calcium deposition was detected at 21–28 days after induction culture by Alizarin Red S staining.