Free-floating long-term vascularized mesenchymal organoids.

The vasculature is essential for tissue function and pathology. Spheroid co-cultures of endothelial and marrow/mesenchymal stromal/stem cells (MSCs) form consistent structures, but the vascular components are short-lived. iPSC-derived vascular organoids can establish complex vasculature but often have variable cell maturation and low reproducibility. This article presents consistently formed, free-floating, long-term vascularized mesenchymal organoids (VMOs), by co-culturing human umbilical vein endothelial cells (HUVECs) and MSCs in a pre-gelled minimal Matrigel scaffold. VMOs support 60-day stable vasculature, exhibiting tissue maturation involving inflammation, extracellular matrix remodeling, and endothelial development. Compared to traditional spheroids, VMOs showed enhanced vascular complexity, sustained extracellular matrix production, and higher cell viability. The system preserved MSC heterogeneity including perivascular cell types, offering physiological relevance. Engraftment of breast cancer cells revealed stromal-tumor niches, enabling modeling of bone marrow metastasis. This robust platform offers an alternative model for studying vascular biology, stromal dynamics, and cancer progression, with potential applications in drug testing.