Drug Loading of Mesenchymal Stem Cells

The drug loading of GinPa-MSCs was performed by priming the cells according to a standardized procedure that use high drug dosage as previously described^{9 (link), 13 (link)}. Briefly, subconfluent cultures (2 × 10⁴ cells/cm²) were exposed to 2 µg/ml PTX, or DXR, or GCB for 24 hours. Then, the cells were washed twice with PBS, trypsinized and washed twice in Hank’s solution (HBSS, Euroclone UK). Drug-primed cells (GinPa-MSCs/PTX, GinPa-MSCs/DXR, GinPa-MSCs/GCB) were then seeded in a 25 cm² flask in DMEM high glucose with 10% FBS and 2 mM L-glutamine (Euroclone, UK) to release the drug. After 48 hours, conditioned media (CM), (GinPa-MSCs/PTX CM, GinPa-MSCs/DXR CM GinPa-MSCs/GCB CM) were collected and tested in vitro for their anti-proliferative activity on SCC154 cells and CFPAC-1 cells (used as standard assay). Conditioned Media from unprimed MSCs were used as control. To determine the amount of drug internalized and not released, cells collected after the releasing phase were lysed by three sonication cycles of 0.4 second pulse cycle and 30% amplitude each (Labsonic U Braun). Lysates from PTX, DXR and GCB primed cells (GinPa-MSCs/PTX LYS, GinPa-MSCs/DXR LYS, GinPa-MSCs/GCB LYS) and unprimed GinPa-MSCs were tested for their anti-proliferative activity.