Chemically-defined medium formulation and adaptation method for supporting growth of endothelial cells.

Chemically-defined (CD) media offer critical advantages for in vitro systems, requiring consistency, tunability, and component transparency, particularly in applications such as bioassays, drug testing, and translational research. While serum-containing media remain widely used, reliable protocols for adapting cells to CD medium, especially for sensitive adherent cell types, are underreported. This study presents a streamlined protocol for adapting human umbilical vein endothelial cells (HUVECs) to a custom CD medium, with a focus on optimizing weaning strategies and identifying surface coatings that support robust attachment and viability. The protocol evaluates gradual and stepwise adaptation approaches to minimize cellular stress, while incorporating defined extracellular matrix proteins to promote adherence under serum-free conditions. Among tested coatings, fibronectin substantially improved cell attachment and viability during CD medium adaptation, outperforming laminin and collagen IV. To support quantifiable and reproducible tracking of cell growth, we applied a trainable AI-based image analysis method for confluence assessment throughout the adaptation process. This work provides a reproducible, modular framework for transitioning HUVECs, and potentially other human cell lines, to CD medium, while preserving cell health and experimental utility across multiple passages. The methodology may also facilitate the design of quantitative and ethically aligned bioassays, accelerating progress in standardized cell culture practices.