Self-assembled Carbon Nanotube/Extracellular Matrix Hydrogel as a Promising Matrix for Organoid Growth and Functional...

Organoids are three-dimensional, self-organized structures derived from tissue-specific cells that recapitulate key architectural and functional features of native tissues in vitro. Current organoid culture systems primarily rely on tumor-derived Matrigel, which suffers from batch-to-batch variability, high cost, and safety concerns, thereby limiting reproducibility and clinical translation. Notably, existing natural and synthetic polymeric hydrogels have not simultaneously supported efficient organoid formation and functional maturation comparable to Matrigel. Here, we present an improved composite hydrogel composed of carbon nanotubes (CNTs) and porcine small intestinal submucosa extracellular matrix (SIS-ECM) as a versatile and biologically active promising alternative matrix for organoid culture. We demonstrate its broad applicability by successfully culturing adult stem cell-derived mouse intestinal organoids (mIOs), human intestinal organoids (hIOs), and human embryonic stem cell-derived vascular organoids (hVOs). Organoids grown within the CNT-SIS-ECM hydrogel exhibited formation efficiency, structural integrity, and functional maturation comparable to, and in certain aspects exceeding, those cultured in Matrigel, underscoring their advantages for transplantation-oriented applications. Mechanistic investigations revealed that CNT incorporation provides instructive bioactive cues that promote organoid development, including upregulation of high mobility group box 1 (Hmgb1), which enhances mIO proliferation and differentiation. Additionally, CNTs augment the WNT signaling pathway, thereby facilitating hVO growth and vascular maturation. Together, these findings establish the CNT/SIS-ECM composite hydrogel as a promising matrix alternative that supports organoid formation and maturation across multiple lineages, offering a practical platform for organoid-based research and tissue regeneration.