Ultra low attachment multiwell plates

Breast cancer cell lines, T47D, BT549, MDA-MB-231 from ATCC, were grown in RPMI-1640 medium. Primary cultures of fibroblasts, deriving from patients treated at the Clinica Mediterranea (Naples, Italy), were grown in Dulbecco's Modified Eagle's Medium/Nutrient F12-Ham (DMEM-F12). Media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. All media and supplements were from Sigma-Aldrich (Milan, Italy).
For mammosphere cultures, single-cell suspensions were plated at a density of 1,000 cells/ml in Corning ultra-low attachment multi-well plates. Cells were grown in stem medium: serum-free DMEM-F12 (Sigma, Milan, Italy) supplemented with B27 (Life technologies, Milan, Italy), 20ng/ml EGF (Sigma, Milan, Italy), 10ng/ml bFGF (BD Biosciences, Milan, Italy), and 1X antibiotic-antimycotics (Life technologies, Milan, Italy). After 4-7 days, mammospheres appeared as spheres of floating viable cells.

Before seeding, all the scaffolds (HA/Gel and Gel scaffolds) were sterilized in 70% v/v EtOH for 2 h, washed two times in PBS 1 × (GIBCO) for 30 min and placed under UV light for 30 min each side. They were then placed in Corning^® ultra-low attachment multiwell plates and conditioned overnight in DMEM/F-12 with 10% serum (i.e. non-differentiating culture medium) in an incubator (5% CO₂, 37 °C). The medium was then discarded and scaffolds ready for seeding. Undifferentiated PDPCs were detached using 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) and seeded at a density of 1 × 10⁴  cell/cm² by applying 50 μL of cell suspension on the samples. After 30 min in an incubator the samples were covered by 1.5 mL of DMEM/F-12 and cultured for 2 and 7 days. The intermediate incubation step was used to ensure the cells adhered to the scaffold without sliding off and falling in the wells.

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)).

A total of 10⁵ cells were transferred to ultralow attachment multiwell plates (Corning) in ESRC (Reprocell, Beltsville, MD, USA) containing 10 ng/ml bFGF, 10 mg/ml human insulin, 100 mg/ml BSA (all from Sigma–Aldrich) and 100 mg/ml human transferrin (Roche, Basel, Switzerland); and incubated as described above for 14 days. The numbers of spheres were manually counted using an inverted microscope (ECLIPSE TE2000-U, Nikon, Tokyo, Japan).