Mesencult expansion kit mouse

Primary ADSCs were isolated from 3- to 4-week-old female CGE transgenic mice. Mouse subcutaneous fat pads were dissected, washed with phosphate-buffered saline (PBS), diced, and dispersed in 2 mg/mL type I collagenase at 37 °C for 30 min. Dulbecco’s modified Eagle medium containing 10% fetal calf serum was then added to stop the enzyme reaction, and undispersed tissue was removed using a cell strainer. The cell suspension was centrifuged at 440× g for 5 min, and the supernatant was removed. Cells were washed with PBS, and supernatant removal via centrifugation, repeated twice. Finally, cell pellets were dispersed with a MesenCult Expansion Kit (Mouse) (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with penicillin–streptomycin–amphotericin B suspension (100×) (FUJIFILM Wako Chemicals, Osaka, Japan), plated on cell culture dishes, and incubated at 37 °C in a humidified incubator with 5% CO₂. Cell suspensions of approximately 1 g of fat pads from five female mice were plated on a 10 cm culture dish.

Osteoblasts were generated from long bones of mice as described previously (Rapp et al., 2018 (link)). Following digestion with 300 U/ml collagenase type IV (Sigma Aldrich) in alpha Minimum Essential Medium (α-MEM; Biochrom, Berlin, Germany) for 2 h with shaking at 37°C and under 5% CO₂ in an incubator, the bones were placed in six-well plates in α-MEM supplemented with 10% fetal bovine serum superior (Biochrom), 1% penicillin/streptomycin, 1% L-glutamine and 0.5% amphotericin B (Thermo Fisher Scientific) in a 37°C and 5% CO₂ incubator. Mesenchymal progenitor cells (MSCs) were isolated from the long bones of mice as described previously (Rapp et al., 2018 (link)). Bone marrow cells were seeded at a density of 5.5×10⁷ cells/cm² in expansion medium (MesenCult™ Expansion Kit, Mouse, Stemcell Technologies, Vancouver, Canada). According to the manufacturer's instructions, the MSCs were cultured with additional MesenPure™ at 37°C and under an atmosphere of 6.0% O₂ and 8.5% CO₂. Osteoblasts and MSCs in passages 3-5 were used for further experiments.