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Methods Mol Biol. 2025 Aug 5. doi: 10.1007/7651_2025_661. Online ahead of print.
ABSTRACT
Although distinguished for their differentiation capacity, human-induced pluripotent stem cells (iPSCs)-derived extracellular vesicles (EVs) have been shown to contribute to functional recovery in the treatment of various traumatic and degenerative diseases. This promising role in therapeutic applications has resulted in considerable attention aimed toward their effective bio-manufacturing. However, traditional culture systems face various insufficiencies. Planar 2D culture results in a lack of scalability, with difficulty in manufacturing clinically relevant doses. Additionally, planar 2D culture lacks the complexity of in vivo biological systems. Although organoids have been proposed to fit this gap by better mimicking in vivo conditions, the traditional generation method of using static culture results in inefficient nutrient and waste transfer. Earlier bioreactor systems, which aim to resolve these issues, also face limitations of homogeneity and stress. Thus, vertical wheel bioreactors (VWBRs) with low shear stress profiles have recently emerged for stem cell organoid cultures, resulting in a more efficient and true-to-form manufacturing process for the secreted EVs. In this chapter, we describe an approach to generate and quantify EVs secreted by iPSC-differentiated human blood vessel organoids (iBVOs) grown in a scalable VWBR. iPSCs are expanded and then differentiated into iBVOs with differentiation media in VWBRs. Their produced EVs are subsequently isolated from the media and quantified using nanoparticle tracking analysis. This culture system should be able to produce a large quantity of the iBVO-derived EVs for the subsequent preclinical and clinical applications.
PMID:40762884 | DOI:10.1007/7651_2025_661