Mesenpro rstm medium

To induce osteocyte differentiation, 5 × 10³ cells/cm² were incubated for 20 days with STEM PRO^® osteogenesis differentiation medium (cat. No. A1007201 Life Technologies). Control cells were cultured in MesenPro RS^TM medium (Life Technologies). Cells were maintained at 37 °C in a 5% CO₂ incubator, and the medium was changed every three days. Following seven days of stimulation, the cells were stained with BCIP/NBT substrate. On day 20, Alizarin Red S staining was performed to assess mineralization.

To induce adipogenic differentiation, 1 × 10⁴ cells/cm2 were seeded in 12-well plates and incubated for 20 days with STEM PRO^® Adipogenesis differentiation medium (cat. No. A1007001 Life Technologies). The adipocyte induction medium was changed every three days. Control cells were maintained in culture in MesenPro RSTM medium (Life Technologies). Following adipogenic stimulation, cytoplasmic lipid droplets were stained with Oil red O. Briefly, the cells were washed with PBS twice and then fixed with pre-cooled 10% formaldehyde for 15 min at −20 °C. After being washed twice with PBS, to remove all the formaldehyde, cells were stained for 30 min at room temperature in freshly diluted Oil Red O Solution 0.5% in isopropanol diluted (3 parts oil red O stock and 2 parts distilled water), filtered with a 0.45 µm filter (O1391-Sigma). After two PBS washes, images were acquired using a bright-field microscope (EVOS M5000 Cell Imaging System, Life Technologies). The percentage and the intensity of the red-stained area were quantified by an ImageJ macro [60 (link)].

Three kinds of hAMSCs were purchased from Invitrogen (Carlsbad, CA, USA), ATCC (Manassas, VA, USA), and Thermo Fisher Scientific, Inc. (Waltham, MA, USA), respectively. The cryopreserved cells were thawed at 37°C and then immediately cultured in MesenPRO RS^TM medium (Gibco, Carlsbad, CA, USA). The cells were then expanded using MesenPRO RS^TM medium to 5 passages. The medium was changed every three days until the cells were 70% confluent, at which time they were passaged.

For cell culture we used Wistar rat adipose-derived mesenchymal stem cells (ADMSCs) purchased from Cyagen Biosciences Inc., Santa Clara, California, USA (Lot Number: 120824L01). The ADMSCs cells were maintained in MesenPRORS^TM medium (Gibco^TM, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin (Sigma-Aldrich, Taufkirchen, Germany), 100 μg/mL streptomycin (Sigma-Aldrich, Taufkirchen, Germany), 2 mM L-glutamine (Sigma-Aldrich, Taufkirchen, Germany) and 1 mmol/l non-essential amino acids (Capricorn Scientific GmbH, Ebsdorfergrund, Germany). Cells were cultivated at 37 °C in an atmosphere of 5% CO₂ and absolute humidity. The culture medium was completely replaced every 3 days until cells reached 80% confluence, after which they were passaged and replated. After the fourth passage (P4), cells were separated from the bottom of the flask by short-acting 0.25% trypsin–EDTA (Gibco, Grand Island, NY, USA). The cells were then resuspended in 10 mL of MesenPRO RS^TM medium to neutralize trypsin and prevent cell damage and then centrifuged at 1500 RPMI for 10 min. Cells were resuspended in 1 mL of MesenPRO RS^TM medium and cell viability was determined using trypan blue staining and only cell suspensions with viability greater than 95% were used.