24 well non adherent plate

OVCAR3 SP cells transfected with hPaf1/PD2 siRNA or Scr siRNA were seeded in triplicates in a 24 well non-adherent plate (Corning Inc., Corning, New York, USA) in CSC-specific media at a concentration of 100 cells/well. The cells in suspension culture were observed under the microscope and fresh media was added every alternate day without removing the existing media. A week later, multiple images were taken per well for different fields of view. The diameter of each tumor sphere was measured using Motic Images Plus 2.0 ML software; the dot plot depicting the diameter of tumor sphere in hPaf1/PD2 knockdown cells and Scr siRNA treated cells was plotted using MedCalc software.

For the purpose of the study, commercial BM-hMSCs (PromoCell, Heidelberg, Germany) were expanded, as previously described [39 (link)]. All experiments were conducted with cells between passage 3 and 4.
Dry scaffolds were placed in a 24-well non-adherent plate (Corning, Sigma-Aldrich, St. Louis, MO, USA) for cell seeding. The cell concentration was adapted to have the required number of cells in a volume of 50μL of growth medium (GM), consisting of DMEM medium supplemented with FBS. A small drop (7 × 10⁵/50 μL viable BM-hMSCs) was slowly deposited on the top of each dry scaffold, waiting for 2 h at 37 °C for the complete absorption of the drop. After this time, 1 mL of GM was added to each well. The cell culture medium was changed three times per week. Each construct was analyzed on day 28 for cell viability and cell proliferation.
Additionally, we seeded BM-hMSCs in the PLA-CH(L), PLA-CH(M) and PLA-CH(H) to investigate the osteogenic differentiation capability of the scaffolds at day 28, following the protocol previously published [39 (link)]. Particularly, BM-hMSCs were grown in osteogenic medium (OM) for 28 days.