Comparative Analysis of Ultra-Low Versus Hydrogel Growing Conditions for Patient-Derived Glioma Organoids

Most data related to high-grade glioma and glioblastoma (GBM) 3D models refer to ultra-low/suspension cultivation systems, for several reasons: easy to initiate, high penetrability of treatments, ease of harvesting for downstream analysis. However, the major limitation of these models is the absence of the extracellular matrix, which enables the specific, local invasiveness of these aggressive brain tumors.

This study provides a comparison between ultra-low and several extracellular matrix (ECM) and hydrogel culture conditions for various grades of patient-derived glioma organoids, in terms of cell growth, invasiveness, and release of cytokines/chemokines, as a model for downstream analysis of soluble factors.

Patient-derived glioma organoids were initiated in three different conditions: ultra-low attachment(UL), 3D Matrigel, and dextran-based hydrogels. Human endothelial cells were used for tube assay co-cultures and human normal monocyte cell line SC to assess monocyte invasion and interaction with tumor organoids.

UL conditions supported organoid formation with better growth rate and easy passaging, and reliable detection of secreted cytokines and chemokines, closely mirroring qualitative secretory profiles observed in tumor explants. In contrast, ECM-based systems enabled invasive growth patterns and co-culture assays, but with a restricted immune cell infiltration and with a reduced detectable level of soluble factors. While qualitative cytokine signatures were preserved across culture platforms, substantial quantitative differences were observed, highlighting the influence of matrix composition on measurable outputs. Preliminary data showed that organoid-forming capacity indicated no clear association with Isocitrate Dehydrogenase 1 (IDH1) mutational status in the tested samples.

Our findings indicate that UL and ECM-based models provide complementary information and recommend a dual-platform cell culture initiation strategy to maximize robustness and translational relevance in patient-derived glioma research.