Abstract
Developing in vitro models to explore cell biology and physiology holds significant importance in biotechnology, cancer research, drug discovery, toxicity testing, and the emerging fields of tissue engineering and regenerative medicine. The conventional two-dimensional (2D) approaches of mammalian cell culture (2D) have limitations in replicating all of the mechanical and biochemical signals in vivo. Culturing cells as spheroids provides a three-dimensional environment that more accurately mimics physiological conditions compared to 2D culture. In this study, we established a method for high-throughput spheroid formation using primary rat hepatocytes (PRHs). Using a high throughput platform, we investigated the effect of varying concentrations of cell culture media supplements on spheroid formation. Additionally, we assessed different cell seeding densities and characterized their properties for 7 days. The average diameter and circularity of PRH spheroids remained stable on days 2, 4, and 7, regardless of the initial seeding density. Moreover, PRH spheroids demonstrated high viability (> 90 %) for up to 7 days across all seeding cell densities. Our results demonstrated that this technique enables straightforward, large scale, consistent, and repeatable spheroid manufacturing and presents an alternative approach for future applications.