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Tissue Engineering

Tissue Engineering - Poster Session D

Poster AA20 - Profiling biomanufactured extracellular vesicles of human forebrain spheroids in a Vertical Wheel bioreactor

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction: Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases including stroke. EVs are membrane-bounded nanoscale particles that carry therapeutic protein and miRNA cargo. However, because of the significant amounts of EVs needed in preclinical and clinical trials, it is still challenging for EV application. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress.

Materials and

Methods: In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells (iPSCs) as 3-D aggregates (Agg) and on Synthemax II microcarriers (MC) in VWBRs was investigated. A static aggregate culture was served as a control. The spheroids were characterized by morphology and metabolites, as well as transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed by proteomics and miRNA-sequencing. The in vitro functional assays using an oxygen and glucose deprived (OGD) stroke model was conducted.

Results, Conclusions, and Discussions:

Results: Human forebrain spheroid differentiated on microcarriers showed higher growth rate than 3-D aggregates. Microcarrier culture had lower glucose consumption per million cells, lower glycolysis gene expression, but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yield from high to low were microcarrier culture, dynamic aggregates, and static aggregates. The cargo enriched with proteins (proteomics) and miRNAs (miRNA-seq) promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability of improving recovery in an in vitro stroke model.

Conclusions: Human forebrain spheroid differentiated in VWBR significantly increased EV yields (up to 240-750 fold) and EV biogenesis compared to static control due to dynamic microenvironment and metabolism alteration. The biomanufactured EVs from VWBRs are enriched with therapeutic cargo and functional in in vitro assays, which paves the way for future in vivo stroke study.

Acknowledgements (Optional):