Model systems play a crucial role in biological and biomedical research, especially in the search for new treatments for challenging diseases like glioblastoma multiforme (GBM). Organoids are 3D in vitro multicellular “middle-ground” model systems that recapitulate highly organized and heterogeneous in vivo organ-like systems, often through stem cell differentiation. Incorporating Matrigel™ or other exogenous ECMs that do not naturally occur in the human body is common practice for organoid generation, ignoring the role of dynamic reciprocity between cells the ECM in tissue development. In this study, we describe a method to develop GBOs from cells without the need for exogenous ECM encapsulation and without cell culture media changes to produce stable tissue-like organoids that reach a 4 mm diameter in as little as six weeks. We observed a transition from homogenous cell populations to tissue-like structures when GBOs were larger than 1 mm diameter. Transcriptomic analysis revealed that the greatest gene expression changes occurred when GBOs were 2 mm in diameter; with collagen VI as the most upregulated ECM-related gene. Quantitative and histochemical assessments further supported native ECM synthesis with significantly higher levels of glycosaminoglycans and collagen in GBOs compared to spheroids. To our knowledge, this study presents the first reproducibly large GBOs with a natively produced extracellular matrices. Organoids with natively synthesized ECMs promise to eliminate artefacts and variability from aged homogenic or xenogeneic scaffolds as well as provide insight for ECM targeted drug development.
