Osteosarcoma-Derived Extracellular Vesicles Induce Lung Fibroblast Reprogramming

Alek Mazumdar et al., Dept. of Orthopedics, Balgrist University Hospital, Zurich, Switzerland; Int. J. Mol. Sci. 2020, 21, 5451; doi:10.3390/ijms21155451

“….our findings provide novel insights into the functional relevance of EV-mediated communication between osteosarcoma cells and normal pulmonary fibroblasts. We deliver strong evidence that osteosarcoma cell-derived EVs can induce lung fibroblast reprogramming and thereby direct fibroblast activation and differentiation towards a myofibroblast/CAF phenotype through EV-associated TGF-beta1 and SMAD2 pathway activation. In conclusion, we highlight osteosarcoma cell-derived EVs as important modulators of the distant microenvironment…” https://www.mdpi.com/1422-0067/21/15/5451

…Materials and Methods

“….Alternatively, to obtain large amounts of EVs from 143-B cells, a bioreactor culture was set-up. A medium-sized, hollow-fiber culture cartridge with a 20 kDa molecular weight cut-o (C2011, Fibercell Systems; Frederick, MD,USA) was pre-cultured according to the manufacturer’s instructions. 143-B cells stably expressing mCherry, luciferase and LacZ [41,42] were expanded in conventional culture flasks and 10^8 cells were used to inoculate the hollow-fiber culture cartridge. Cells were then slowly adapted to bioreactor culture conditions over two weeks by gradually increasing the concentration of protein-free medium (1:1 DMEM/Ham’s F12 + 10% Fibercell Systems chemically defined medium for high density cell culture (CDM-HD; Fibercell Systems, Frederick, MD, USA) protein-free supplement + 100 U/mL penicillin/streptomycin). Bioreactor-conditioned medium (20 mL) was collected for each harvest 3–5 times per week depending on the glucose concentration of the medium reservoir. Harvests were precleared of cells and debris by sequential centrifugation at 300 g for 10 min and 2500 g for 15 min…”