Mscgm cd mesenchymal stem cell bulletkit

The hMSCs used in this study were limited to 3–5 passages in order to match the conditions in which hMSCs are used in the clinical setting. hADSCs were cultured on a 100 mm culture plate using a chemically defined medium (CDM) (MSCGM-CD mesenchymal stem cell BulletKit (Lonza 00190632)) (the number of cells was 3 × 10⁶/plate) until reaching 80% confluence. After 48 h, the culture supernatant was aspirated with a pipette and centrifuged (1500× g, 30 min, 4 °C) to remove the cells. After centrifugation of the medium, the supernatant was concentrated 20 times using Amicon Ultra-15, PLGC Ultracell-PL membrane, 10 kDa (MERCK UFC 901008)(Merck Millipore, Darmstadt, Germany), after which the component proteins were analyzed by LC–MS/MS.

hADSCs (46-year-old Caucasian female) (PromoCell, Heidelberg, Germany) were cultured (37 °C, 5% CO₂) on a coated 100 mm culture plate (TPP 93100). Then, after reaching 80% confluence passage of cells was performed every 3–4 days. The cells were washed with Phosphate buffered saline (PBS) (calcium, magnesium-free), and hADSCs were dissociated using a dissociation solution. Subculturing was carried out by plating on uncoated 100 mm culture plate. An MSCGM-CD mesenchymal stem cell BulletKit (Lonza 00190632) or DMEM-10% FBS was used for the culture medium. Trypsin/ethylenediaminetetraacetic acid (EDTA) (Lonza CC-3232) was used for the dissociation solution.