Background: Gliomas exhibit significant heterogeneity in drug responses, necessitating precise in vitro models to evaluate chemotherapeutic efficacy. Conventional 2D cultures fail to recapitulate the three-dimensional tumor microenvironment and dynamic drug exposure profiles. Objective: This study aims to develop a microfluidic chip capable of generating stable chemotherapeutic concentration gradients for high-throughput evaluation of drug sensitivity in three-dimensional glioma spheroids. Methods: Glioma cells (U87) were cultured into uniform spheroids (~200 µm diameter) and loaded into a microfluidic device designed with a concentration gradient generator. Temozolomide (TMZ) was applied in a linear gradient ranging from 0 to 500 µM. Cell viability was assessed after 72 hours by Calcein-AM/PI staining and quantified via fluorescence microscopy. IC50 values were calculated based on dose-response curves. Reproducibility was biologically evaluated by triplicate experiments. Results: The microfluidic platform generated a stable TMZ gradient with <5% concentration variance across channels. Cell viability decreased dose-dependently with an IC50 of 220 ± 15 µM. At 500 µM TMZ, viability was reduced to 28 ± 4%, whereas at 50 µM, viability remained above 80%. Spheroids exhibited heterogeneous responses, reflecting diverse tumor cell phenotypes. The system demonstrated high reproducibility (R² = 0.98) and sensitivity in detecting subtle drug effects. Conclusion: This microfluidic drug sensitivity chip offers a physiologically relevant and reliable platform for assessing glioma chemotherapeutic responses under dynamic concentration gradients. The approach enables personalized drug screening and may accelerate therapeutic optimization in glioma treatment.

Glioma, Microfluidic Chip, Drug Sensitivity, Concentration Gradient, Spheroid Model, Temozolomide