014 | Development of a standardized and reproducible workflow for the generation and characterization of patient-derived glioblastoma spheroids

Martina Di Marco¹, Alessandra Maria Vitale¹, Giuseppa D’Amico¹, Alessandro Lo Giudice¹, Francesca Chiara Cecala¹, Francesco Cappello^1|2, Celeste Caruso Bavisotto^1|2 | ¹Institute of Human Anatomy and Histology, Department of Biomedicine, Neurosciences and Advanced Diagnostics, University of Palermo, Italy; ²Euro-Mediterranean Institute of Science and Technology IEMEST, Palermo, Italy.

Glioblastoma (GBM) is a highly aggressive and therapy-resistant primary brain tumor, largely driven by invasive behavior and pronounced intratumoral heterogeneity, which encompasses genetic, epigenetic, phenotypic, and functional diversity among tumor cell subpopulations [1]. Traditional two-dimensional (2D) monolayer cultures fail to adequately recapitulate these features, as they impose artificial growth conditions that profoundly alter cell morphology, polarity, metabolism, and response to therapies [2]. In recent years, patient-derived three-dimensional (3D) culture models, including tumor spheroids, have emerged as more physiologically relevant systems that preserve key features of GBM ex vivo, such as cellular diversity, phenotypic plasticity, and microenvironmental interactions [3]. Despite their relevance, standardized and reproducible protocols for the generation of GBM spheroids from surgical specimens remain limited. This study describes a robust and standardized methodological workflow for the isolation, 3D cultures, and expansion of primary GBM cells obtained from patient biopsies, enabling the reproducible formation of stable and morphologically consistent spheroids. The protocol includes step-by-step procedures for tissue dissociation, cell seeding under low-adhesion conditions, optimization of culture density, and long-term spheroid maintenance. In addition, guidelines for the immunomorphological characterization of the resulting 3D structures are provided. This workflow establishes a reliable in vitro platform for GBM modelling and supports downstream applications such as tumor biology studies, drug screening, biomarker validation, and patient-specific therapeutic testing in a three-dimensional context.