A 3D Astrocyte Microenvironment Model Enables Rapid Ca 2 + ‐Resolved Analysis and Therapeutic Modulation of Oxidative Neuroinflammation

ABSTRACT Astrocytes orchestrate key neuroinflammatory processes, yet conventional two dimentional (2D) cultures distort Ca 2+ signaling and elevate inflammatory noise. Here, we introduce the Astrocytic Reactive‐Calcium 3D microenvironment (ARC‐3D), a tunable methacrylated gelatin (GelMA)‐based 3D astrocyte microenvironment that enables stable long‐term culture, improved transcriptional fidelity, and rapid Ca 2+ ‐resolved functional assessment under perfusion. The optimized 3.5 wt% matrix supported quiescent, structurally mature astrocytes and markedly reduced the inflammatory baseline observed in 2D systems. Upon oxidative stimulation with H 2 O 2 , ARC‐3D recapitulated hallmark features of reactive astrogliosis, including elevated basal Ca 2+ levels, impaired receptor‐evoked Ca 2+ transients, induction of GFAP and MAO‐B, and secretion of IL‐6 and IL‐1β, captured at second‐to‐minute timescales. Treatment with KDS12025, an H 2 O 2 ‐scavenging MAO‐B modulator, partially restored Ca 2+ homeostasis, suppressed Ca 2+ ‐dependent cytokines, and selectively recovered gliogenic and metabolic signatures in transcriptomic analysis. In vivo intracerebroventricular H 2 O 2 challenge validated the ARC‐3D pathological trajectory and therapeutic modulation, highlighting strong in vitro–in vivo concordance. Collectively, ARC‐3D offers a mechanistically precise, Ca 2+ ‐resolved, and translationally aligned platform for decoding oxidative neuroinflammation and for advancing astrocyte‐targeted therapeutic strategies.