Mesenpro rs growth supplement

ADSCs (Invitrogen) were re-expanded in MesenPRO RS Basal Medium (Invitrogen) and supplemented with MesenPRO RS Growth Supplement (Invitrogen), 1% Penicillin/Streptomycin (Invitrogen), 1% L-glutamine (Invitrogen, USA), and 1% AmphotericineB (Invitrogen). Cells were maintained in a humidified incubator at 37°C in 5% CO₂–95% air atmosphere and new media every second day. Cells from the third passage were used for tissue spheroid biofabrication.

Human adipose-derived MSCs were purchased from Invitrogen (Carlsbad, CA, USA). MSCs were cultured in Mesen PRO RS Basal Medium supplemented with Mesen PRO RS growth supplement (Invitrogen) and incubated at 37℃ in a 5% CO₂ atmosphere. Cells were used after 2-5 passages. MSCs were labeled using the QDs (Q-Tracker 800) Cell Labeling Kit (Invitrogen). MSCs (1×10⁶) were suspended with 200 µL of complete growth medium and labeled with 10 nM of QDs labeling solution. After 60 minutes, labeled cells with QDs were washed twice with complete growth medium.

Human adipose-derived stem cells (ADSCs) were purchased from the Invitrogen Technologies and the cells from three donors were mixed and expanded to reach passage four by culturing in MesenPRO basal medium with 2 mM l-glutamine and MesenPRO RS Growth Supplement (Invitrogen). Passage 4 ADSCs were used for all the experiments. The attachment of ADSCs cultured for 24 h on the ceramic disks was analyzed using scanning electron microscopy. Briefly, the ADSCs were seeded on the disks at the density of 10,000 cells per mL and cultured for 24 h in growth medium consisting of α-minimal essential medium (α-MEM, Gibco Laboratories), supplemented with 10% (v/v) foetal calf serum (Gibco Laboratories), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco Laboratories). After 24 h of culture, cells were rinsed in phosphate-buffered solution (PBS), fixed in 1.25% glutaraldehyde and sequentially dehydrated in graded ethanol. Samples were dried in hexamethyldisilazane and coated with gold for scanning electron microscopy (SEM) analysis using FE-scanning electron microscopy (Zeiss Ultra).

To study biological activity of the scaffolds, human adipose-derived stem cells (hADSCs) were commercially purchased (ATCC,UK) and cultured in MesenPRO RS™ basal cell culture medium (ThermoFisher, UK) supplemented with 2% MesenPRO RS™ growth supplement (ThermoFisher, UK) and 1% penicillin/streptomycin (Sigma-Aldrich, UK) at 37^ᴏC with 5% CO₂. All the in vitro cell based experiments were carried out at passages three or four and scaffolds were seeded with 7.5 × 10⁵ cells.

The StemPro™ human adipose-derived mesenchymal stem cell line (R7788115) was obtained from Thermo Fisher (Waltham, MA, USA). The human adipose-derived mesenchymal stem cells (hADMSCs) were cultured in MesenPRO RS™ Basal Medium supplemented with MesenPRO RS™ Growth Supplement (12,746,012, ThermoFisher) in a controlled incubator with a humidified atmosphere containing 5 % CO₂ at 37 °C. Sub-culturing was performed when the cell culture reached 70 % confluency, and cells at passage 8 were used for the experimental procedures.