Loading GinPaMSCs with Paclitaxel

To load GinPaMSCs with PTX, the cells were primed with a high amount of drug according to a standardized procedure previously described [7 (link),15 (link),17 (link)]. Briefly, cultures were obtained by seeding 2 × 10⁴ cells/cm², and after 72 h, cells were exposed to 2 µg/mL PTX for 24 h. Then, after washing twice with phosphate buffered saline (PBS), the cell monolayer was trypsinized, washed in Hank’s solution (HBSS)( Euroclone, Pero, Italy), and the PTX-primed cells (GinPaMSCs/PTX) were seeded in a 25 cm² flask in DMEM HG with 10% FBS and 2 mM l-glutamine (Euroclone, Pero, Italy) to release the drug. After 48 h of incubation into conditioned media (CM), PTX-loaded GinPaMSCs (GinPaMSCs/PTX/CM) were collected and tested in vitro for their anti-proliferative activity on different tumor cell lines (see Section 2.7). In particular, human pancreatic adenocarcinoma cell line CFPAC-1 was used as a standard laboratory assay according to the method reported below. CM from untreated MSCs were used as control.