Abstract
INTRODUCTION: Three-dimensional (3D) human brain models have become indispensable tools to investigate neuroimmune interactions and inflammatory processes in the human central nervous system in vitro. Nevertheless, existing models, including brain organoids and other iPSC-derived systems, are often constrained by lengthy differentiation protocols, considerable cost, and substantial batch-to-batch variability, restricting their applicability in translational neuroimmunology. METHODS: We developed a scalable and reproducible 3D human neurospheroid model (tri-hNSPHs) composed of neuronal, astrocytic, and microglial human cell lines, specifically designed to study neuropathogenic mechanisms. Tri-NSPHs were exposed to a defined pro-inflammatory cytokine cocktail (IL-1β, TNFα, and IFNγ) to quantify the secretion of multiple inflammatory mediators. The inflammation was also counteracted using distinct anti-inflammatory pharmacological compounds and cellular adaptations to hypoxic stress were modeled. RESULTS: Tri-hNSPHs rapidly self-assemble while maintaining key neuro-glial interactions and enabling precise analysis of immune responses not attainable with conventional two-dimensional cultures. The stimuli we provided triggered robust and quantifiable inflammatory activation, demonstrating the versatility of the model and its suitability for dissecting neuroinflammatory pathways. Pharmacological modulation effectively attenuated these responses, further validating the platform for mechanistic and therapeutic studies. In addition to modeling neuroinflammation, tri-hNSPHs reliably recapitulated cell reactions to hypoxic stress, a pathological condition tightly intertwined to neuroimmune activation in numerous neurological disorders. DISCUSSION: Together, these findings establish tri-hNSPHs as a scalable, experimentally robust, and translationally relevant 3D neuroimmune model for investigating inflammation-driven brain pathology and evaluating anti-inflammatory strategies in a controlled and reproducible in vitro setting. This platform holds significant promise for advancing neuroimmune research and preclinical screening of immunomodulatory therapies.