Exploring Integrin Function to Enable Defined, Animal-Free Organoid Culture: From fundamental insights to reproducible, simple human organoid systems through integrin-guided matrix design

Abstract

The discovery of human and murine adult Lgr5⁺ stem cells (ASCs) has enabled the development of organoids that faithfully recapitulate the architecture and function of epithelial tissues derived from all three germ layers. These self-organizing “mini-organs” are genetically stable, contain tissue-specific cell types, and can be expanded long-term in vitro using organ-specific growth factor cocktails. Such media typically contain four key components: Wnt pathway agonists to maintain LGR5⁺ stem cells, EGF-type growth factors to stimulate proliferation, TGF-β/BMP inhibitors to prevent differentiation, and an extracellular matrix (ECM) to provide essential three-dimensional structural and biochemical cues via integrin-mediated signaling. By optimizing these growth factor combinations, diverse human organoid types have been established from healthy and diseased tissues. Which makes the organoid technology a powerful system for studying human epithelial biology, modeling disease, and evaluating personalized therapeutic responses. Genetic modification further extends the platform’s utility for modeling hereditary disorders, repairing gene defects ex vivo, and exploring regenerative medicine applications and transplantations. Despite major advances, a key bottleneck remains the reliance on Matrigel, as the ECM component for organoid culture. Its undefined composition, batch variability, and tumor origin hinder reproducibility and clinical translation. Understanding the integrin-mediated mechanisms that govern cell–matrix interactions is therefore crucial for developing defined, synthetic ECM alternatives. This dissertation investigates the role of β1 integrins and their associated α-subunits in organoid adhesion and growth. We identify the integrin-activating antibody TS2/16, originally described in immune studies, that promotes epithelial organoid expansion in the presence of ECM proteins. In addition, the integrin-binding domain of bacterial protein invasin is shown to activate critical epithelial integrin receptors, enabling organoid growth in both two- and three-dimensional culture systems. Together, these findings advance our understanding of integrin functionality in organoid biology and support the development of defined and animal-free organoid culture systems suitable for translational and clinical applications.