The creation and validation of a fully animal component-free media for select adherent cell types

Fetal Bovine Serum (FBS) is one of the most commonly used media supplement for the maintenance of mammalian cell types, yet the expensive costs, ethical concerns, and lot-to-lot variation have provoked a clear need for a serum that is standardized and derived from non-animal sources. Several serum-free formulations have been developed in the past, however they are often cell type specific, contain animal-derived components, and lack long-term culture validation. In this study, we developed a novel animal component-free (ACF) media and investigated its effectiveness on four commonly used mammalian cell lines via long-term (up to 90 days) morphological, transcriptomic, and proliferative analyses. Cells cultured in our ACF medium exhibited comparable cellular morphologies and equal or greater growth rates compared with cells cultured with FBS. Additionally, differentially expressed genes between the FBS-grown and ACF-grown groups were predominantly associated with functions linked to proliferation and cell attachment. While the tested cells were initially derived using conventional methods and include non-human lines, this study demonstrates that our medium supports long-term culture without animal-derived supplements. The findings from this study indicate that this medium is a suitable replacement to FBS-containing medium for several common cell lines. Insight Box Traditional cell culture methods often rely on animal-derived components, which can pose ethical and economic challenges. The use of animal serum in vitro is needed to supply nutrients to cells but raises concerns about animal welfare and introduces variability and contaminants that can negatively affect downstream applications. This study presents a novel animal component-free medium designed to support the growth of adherent cell types, providing a sustainable alternative to serum. Here, we demonstrate long-term cell viability, normal morphology, and differential gene expression patterns indicative of enhanced proliferation and attachment in cells cultured in 2D environments. By addressing the demand for ethical and reproducible cell culture methods, this research aims to contributes to the broader adoption of sustainable practices in biotechnology.