Biomanufacturing and lipidomics analysis of extracellular vesicles secreted by human blood vessel organoids in a vertical wheel bioreactor.

Abstract

Background: Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However, the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs), with unique design features, facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress, using aggregate- and microcarrier-based culture systems.

Methods: Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling, flow cytometry, and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blotting. Lipidomics provided insights into EV lipid composition, while functional assays assessed the impact of EVs in a D-galactose-induced senescence model.

Results: VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size, but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however, distinct differences were identified in lipid chain lengths and saturation levels, affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro.

Conclusions: Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2-3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo, showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential, paving the way for future in vivo studies.