Slovackova, J.; Bernatik, O.; Cimborova, K.; Barak, M.; Hendrych, M.; Kocourkova, K.; Sulcova, M.; Olha, J.; Amruz Cerna, K.; Hodny, Z.; Jancalek, R.; Bohaciakova, D.

Background: Patient-derived tumor organoids are widely used in cancer research, yet the biological impact of tissue processing during model generation remains unclear. Fragment-based and dissociation-based approaches are commonly assumed to trade fidelity for uniformity, but their molecular consequences remain incompletely defined. Methods: We performed a proteome-wide comparison of fragment-based (CUT) and dissociation-based (DIS) glioblastoma organoid protocols using quantitative mass spectrometry. Organoids from multiple patient tumors were cultured under growth factor-free or growth factor-supplemented conditions and compared with matched primary tissue. Results: Both protocols produced technically robust glioblastoma organoids when maintained in their native media. However, CUT organoids matched the reproducibility of DIS cultures while preserving a broader extracellular matrix repertoire and networks linked to collagen assembly, vascular support, and cel-matrix signaling. DIS cultures were biased toward exogenous basement membrane components and proliferative, growth factor-responsive states. Across tumors, CUT organoids consistently showed greater proteomic similarity to matched primary tissue, retaining neural, glial, stromal, and extracellular features largely absent from DIS models. Conclusions: Fragment-based glioblastoma organoids can be both reproducible and biologically faithful. Tissue dissociation acts as a major perturbation that reshapes extracellular matrix organization, cellular states, and tumor identity, making protocol choice a critical determinant of model fidelity and translational relevance.