Small RNA Deep Sequencing of Vesicles released by LIM1863 Cell Line. Homo sapiens strain:LIM1863

For large scale production of exosomes, cells were cultured in CELLine Bioreactor classic flask (Integra Biosciences, Switzerland) and culture medium (CM) collected. Approximately 3x107 LIM1863 cells were suspended in 15 ml of phenol red free RPMI 1640 medium supplemented with 0.5% ITS, 100 U/mL penicillin and 100 ¦Ìg/mL streptomycin (cultivation medium) and seeded into the cultivation chamber of a CELLine classic flask. The nutrient supply chamber of the CELLine classic flask was filled with 500 ml of RPMI1640 containing 5 % FCS, 100 U/mL penicillin and 100 ¦Ìg/mL streptomycin. The cell culture medium in the nutrient supply chamber was replaced twice a week and the cell suspension in the cultivation chamber was collected every 48 h. After each collection, the cell suspension was centrifuged at 250 ¡Á g for 3 min to sediment organoids CM. The organoid pellet was resuspended in 15 ml of cultivation medium and re-seeded back to the cultivation chamber of the flask. CM was centrifuged at 2,000 ¡Á g to remove floating cells and cell debris followed by centrifugation at 10,000 x g for 30 min at 4oC to collect shed sMVs. The resultant supernatant was centrifuged at 100,000 x g for 1 h at 4oC for isolating crude exosomes. The crude exosome pellets were stored at -80 oC for immunocapture exoperiments.