A viscoelastic suspension culture strategy modulating fusion and development in blood vessel organoids

Human blood vessel organoids (hBVOs) demonstrate significant potential in vascular drug development and tissue repair engineering. However, the traditional liquid culture method leads to spontaneous cell fusion and lacks a defined mechanical microenvironment to support cell growth during the initial suspension culture process for hBVOs, which limits their homogeneity under large-scale cultivation and the stability of their angiogenic properties. This study developed a novel viscoelastic culture medium with dual mechanical functions to replace the conventional liquid culture medium. By adding xanthan gum, we constructed a "rigid" barrier that could resist the transient stress generated under external disturbances, thereby inhibiting the contact and fusion of hBVOs. Concurrently, the system exhibits stress-relaxing "soft" properties in response to long-term stresses arising from hBVOs growth and expansion, providing appropriate mechanical cues while supporting cell growth. Our approach elevated hBVOs residual proportion from 31.35% to 84.23%, while significantly reducing size coefficient variation from 0.53 to 0.21. Furthermore, this viscoelastic medium can promote the densification and pre-vascularization of the internal tissues of hBVOs. Compared with traditional liquid culture hBVOs, after embedding them in type I collagen gel, they exhibit a stronger angiogenic ability, with the total length of blood vessels increasing by 124.1%. Transcriptomic analysis further confirms that this environment upregulates pathways related to mechanotransduction and angiogenesis. This study not only provides a novel strategy for the efficient, standardised preparation of hBVOs but also offers fresh perspectives for research into mechanically regulated vascular development within microenvironments.