Fig. 2

Monitoring of culture parameters during the hollow-fiber bioreactor incubation period demonstrates feasibility and consistency of cell homeostasis. a Graphical schematic representation of the entire bioreactor-based workflow for the production of extracellular vesicles from hBM-MSCs in the hollow-fiber bioreactor system. A 7-day preparation period of the hollow-fiber cell culture system was required before cell inoculation where PBS was injected for 5 days followed by a 2-day incubation with RoosterBio complete cell culture medium. At day 7, hBM-MSCs were inoculated into the hollow-fiber cartridge. The medium in the extracellular capillary space (ECS) was replaced by the RoosterCollect-EV medium for 2 days during this 3-day period of cell inoculation. At day 10, the 25-day EV production period started and 20 mL of the EV-rich cell-conditioned medium was retrieved daily from the ECS and frozen for future EV isolation and analysis. Aliquots of EV-rich cell-conditioned medium harvested at early (days 1–2), interim (days 13–14), and end (days 24–25) of production time points were selected for further EV downstream analysis. The glucose and lactate concentrations were measured every 2–3 days. At the end of the 25-day EV production period, hBM-MSCs were harvested from the cartridge and analyzed for MSC features. b Glucose concentration (i) in the circulating medium was monitored at a 2–3-day interval schedule to ensure consistent glucose consumption by cells. Indicated by the inflections between days 16 and 18 is representative of glucose replenishment by adding fresh cell culture medium. L-Lactate production was assessed using circulating media samples by measuring L-Lactate concentrations (ii) using a colorimetric plate-based assay to ensure cells did not achieve metabolic stress (N = 4 donors; hBM-MSC-48RB/81RB/55RB/85RB) (N = 2 technical replicates)