A 3D Astrocyte Microenvironment Model Enables Rapid Ca² ⁺-Resolved Analysis and Therapeutic Modulation of Oxidative Neuroinflammation.

Abstract

Astrocytes orchestrate key neuroinflammatory processes, yet conventional two dimentional (2D) cultures distort Ca²⁺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 Ca²⁺-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 H₂O₂, ARC-3D recapitulated hallmark features of reactive astrogliosis, including elevated basal Ca²⁺ levels, impaired receptor-evoked Ca²⁺ 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 H₂O₂-scavenging MAO-B modulator, partially restored Ca²⁺ homeostasis, suppressed Ca²⁺-dependent cytokines, and selectively recovered gliogenic and metabolic signatures in transcriptomic analysis. In vivo intracerebroventricular H₂O₂ challenge validated the ARC-3D pathological trajectory and therapeutic modulation, highlighting strong in vitro-in vivo concordance. Collectively, ARC-3D offers a mechanistically precise, Ca²⁺-resolved, and translationally aligned platform for decoding oxidative neuroinflammation and for advancing astrocyte-targeted therapeutic strategies.