Optimization of Tumor Spheroid Preparation and Morphological Analysis for Drug Evaluation

Due to its similarity to in vivo conditions, tumor spheroids are actively used in research areas, such as drug screening and cell–cell interactions. A substantial quantity of spheroids is crucial for obtaining dependable results in high-throughput screening. Conventional fabrication methods of spheroid have limitations in low yield and morphological variation. Dropletbased microfluidic system capable of mass-producing uniformed spheroids can overcome these limitations. In this study, we investigated the optimal culture conditions, which allows to researchers provide guidelines for producing spheroids with the desired diameter and quantity. Mass-produced spheroids were employed to analyze compaction, which is crucial for evaluating the remission effects of drugs, as well as the formation of a necrotic core, which induces a bias in the analysis of drug response and viability. The time point at which compaction is completed and the diameter begins to increase was measured using spheroids with diameters of both > 400 μm and < 400 μm, and spheroids do not proliferate a linear growth trend. Spheroid with diameters ranging from 73.4 ± 11.42 μm to 371 ± 5.11 μm was used to predict the formation of the necrotic core based on live cell counting, and diameter of 300–330 μm was mathematically calculated as the diameter where a necrotic core forms. Additionally, the use of artificial intelligence (AI) for high-throughput analysis is crucial for obtaining time-saving and reproducible data. We produced BT474 and MCF-7 spheroids with diameters of 100, 200, and 300 μm and obtained morphological indicators from an AI-based program to compare the differences in heterogeneous breast tumor spheroids. Through this study, we optimized the diameter of spheroids and the initiation timing for drug screening and emphasized the importance of AI-based morphological analysis in high-throughput screening.