Time-dependent cytokine landscapes in an ex vivo microfluidic glioblastoma platform

Glioblastoma (GBM) is the most common and aggressive astrocytic glioma of the central nervous system with a median survival of 15 months from diagnosis. Patient outcomes have improved only marginally over the past 20 years, and GBM research is limited by models that fail to preserve the native architecture and function of tumours over time. The aim of our study was to provide a physiologically and clinically relevant microfluidic environment for the study of GBM. We designed and validated a custom-built polymethyl methacrylate (PMMA) microfluidic perfusion platform, and successfully maintained ex vivo culture of intact, fresh, patient-derived GBM biopsies for up to 12 days. Cellular viability and tissue integrity were assessed by histology, apoptosis markers, and cytokine profiling, whilst cellular stress was quantified by lactate dehydrogenase (LDH) release. LDH levels initially decreased and then remained low and stable up to 12 days. Haematoxylin and eosin staining showed that tissue architecture was preserved, with mitotic figures post-perfusion. Cleaved PARP and Annexin V immunohistochemistry revealed no significant increase in apoptosis between pre- and post-perfused tissues at 8 or 12 days. Effluent cytokine analyses demonstrated sustained secretory activity with a time-dependent modulation (across 105 analytes: p = 0.0288). Quantitative ELISAs for seven cytokines (VEGF, MMP9, CHI3L1, IL-6, IL-8, Serpin-E1, Angiopoietin-2) corroborated cytokine analysis trends and correlated strongly with array data (p < 0.001), validating assay fidelity and biological signal. Collectively, biochemical, histological and secretome readouts show that intact GBM tissues remain viable, metabolically active and analysable on this platform for up to 12 days. The system provides a practical, physiologically relevant, model for longitudinal GBM studies and lays the groundwork for scalable translational testing of patient tissue.