Real-time monitoring and quantitative analysis of 3D tumor spheroids using portable cellular imaging system.

Three-dimensional (3D) tumor spheroid models closely mimic in vivo tumor environment and play a vital role in studying oncological research. Despite their significance, the existing methods for analyzing 3D tumor spheroids often suffer from limitations, including low throughput, high cost, and insufficient resolution. To address these challenges, we developed a portable imaging system for the real-time sensing and quantitative analysis of the 3D tumor spheroids. The system integrated the seamless workflow of spheroid generation, cell morphology tracking, and drug screening. The spheroid generation was successfully characterized using MCF-7 breast cancer cells by optimizing cell concentration (5-20 × 10(6) cells/mL), incubation time (24-96 h) and microwell diameter (400-600 μm). A custom-written algorithm was developed for automated analysis of spheroids, exhibiting high sensitivity (98.99%) and specificity (98.21%). Confusion matrices and receiver operating characteristic curve analysis further confirmed the robustness of the algorithm with an area under the curve value of 93.75% and an equal error rate of 0.79%. Following the characterization, the real-time sensing of spheroid generation and the response of spheroids to drug treatment were successfully demonstrated. Furthermore, the live/dead assays with chemotherapy provided a detailed insight into the efficacy and cytotoxic effects of the drug, demonstrating a significant dose-dependent decrease in a spheroid viability. Therefore, our system offers considerable potential for enhancing drug development processes and personalized treatment strategies, thereby contributing to more effective cancer therapies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13534-025-00470-y.