(277b) Biomanufacturing and Lipidomics Analysis of Extracellular Vesicles Secreted By Human Blood Vessel Organoids in a Vertical Wheel Bioreactor

Extracellular vesicles (EVs) produced by human brain cells primarily for cell-to-cell communication have been shown to be a promising cell-free therapeutic in various diseases including senescence, neurodegeneration, and stroke. EVs are phospholipid bi-layer bound particles on the nanoscale that hold therapeutic protein and miRNA cargo. Due to the vast quantities of EVs needed for preclinical and clinical trials, it is still challenging for EVs to be utilized due to their low harvesting yield. 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. There is limited research on the effects of aggregate- or microcarrier-based organoid culture systems within a dynamic culture environment. There is also limited research on how the upscaling of organoid generation in a VWBR can affect cell quantity, EV production, and lipid composition of EVs. This study addresses this oversight in current research.

In this study, the EV secretion by human BVOs derived from induced pluripotent stem cells as 3-D aggregates and on Synthemax II microcarriers in VWBRs at varying levels of shear stress (~0.4 vs. 0.2 dyn/cm² on average) were investigated. A 3-D static aggregate culture was served as a control. The organoids were characterized for morphology, growth kinetics, and metabolites. Flow cytometry, immunohistochemistry, and gene analysis were performed to assess differences in differentiation efficiency between culture conditions. Spent media were collected and the EVs were isolated via an ExtraPEG ultracentrifugation process. The isolated EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot. The EV cargo was analyzed by lipidomics. for differences in lipid content, composition and Kyoto Encyclopedia of Genes and Genomes (KEEG) pathway enrichment. An in vitro functional assay of a D-galactose induced senescence model was conducted to assess the therapeutic potential of the generated EVs.

Human BVOs differentiated on microcarriers showed higher growth rate than as 3-D aggregates. VWBR cultures had more aerobic metabolism, lower glucose consumption per million cells, higher glycolysis gene expression, and higher EV biogenesis genes when compared to the static control. EVs from different culture conditions showed no differences in size, but the yields from high to low were microcarrier culture under high shear stress, microcarrier culture under low shear stress, dynamic aggregates under high shear stress, dynamic aggregates under low shear stress, and static aggregates. The EV lipidomics revealed regardless of culture condition (static vs. dynamic and aggregates vs. microcarrier) total lipid content was similar as well as the subclass lipid composition, with variations less than 0.36%. Heatmap revealed each culture condition however had distinct subclass compositions. Within subclasses, there are statistical differences between abundance of chain lengths and saturated molecules between cultures. Volcano plots revealed upregulated (23) and downregulated (24) lipids between each culture condition and the top 20 pathways (e.g., Sphingolipid signaling, Neurotrophin signaling) affected by these differences were identified. Human BVO EVs demonstrated the ability of reducing reactive oxygen species (ROS) and increasing cell proliferation of an in vitro senescence model.

Human BVOs differentiated in VWBRs (in particular low shear stress) 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. EV lipidomics revealed that VWBR samples had lower concentration of unsaturated lipids per subclass, therefore having higher rigidity. Sphingolipid metabolism and signaling pathways are the most prevalent mechanisms in VWBR samples that differ from the static control. 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.