eng Oriental Scientific Publishing Company Biomedical and Pharmacology Journal 0974-6242 2025-06-30 18 2 1610 1620 10.13005/bpj/3198 66065 article Wenyan Xu 1 Bo Zou 1 Guohao Xie 2 Yeqing Wu 2 H. Fai Poon 2 School of Medicine, Sun Yat-Sen University, Shenzhen, China. Research and Development Department, Quacell Biotechnology Co., Ltd., Zhongshan, China. Cell culture plays a pivotal role in the biomedical industry, with a growing shift from traditional serum-containing adherent systems to serum-free suspension culture platforms. This transition underscores the importance of developing robust cell suspension adaptation protocols and serum-free culture media to meet industrial demands. In this study, we employed a combination of cell suspension acclimation, differential screening of culture media, and factorial design methodology to rapidly adapt adherent human embryonic kidney (HEK293) cells to suspension culture. Additionally, we developed a chemically defined medium optimized for these cells within a short timeframe. By systematically adjusting the concentrations of key medium components and integrating an optimized adaptation protocol, we successfully achieved efficient suspension culture of HEK293 cells. Notably, this approach significantly reduced cell aggregation and enhanced cell growth density, thereby advancing the potential for industrial-scale applications. https://biomedpharmajournal.org/vol18no2/development-of-serum-free-medium-and-suspension-culture-for-hek293-cells-using-systematic-optimization-strategies/ Cell adaptation HEK293 cells Medium optimization Plackett - Burman experiment Serum-free suspension culture