A New Sustainable Approach: Generation of a Serum-Free Cell Line for Enhanced Bioassay Applications

In vitro cell-based methods are essential for characterizing molecules and conducting potency bioassays for clinical and marketed drugs, as required by regulatory authorities. Fetal bovine serum (FBS) has been considered the gold standard for cell supplements, offering hormones, lipids, and growth factors that mimic the natural cellular environment. However, concerns about the variability and undefined nature of FBS compromise the reliability and consistency of scientific results. Additionally, FBS serves as contamination risks and raises ethical issues regarding animal-derived products. Eliminating FBS from cell culture processes presents a safe, cost-effective, and ethical alternative for pharmaceutical companies. This project aims to adapt HEK-293T cells to a serum-free (SF) environment and establish SF cell banks for bioassay development. Two formulations of serum-free media (SFM) were developed, and various adaptation protocols were tested. A robust protocol was defined, allowing for the adaptation of HEK-293T cells within one week in SF conditions. Two Master Cell Banks were generated, followed by a characterization study that confirmed the stability of cell lines over time, demonstrating consistent viability, proliferation, morphology, and cell cycle characteristics. To assess the suitability of HEK-293T SF cells for bioassay development, cells were transfected to express specific molecule targets using lipid-based reagents, electroporation, and viral transduction. Lipid-based reagents and viral transduction showed promising transfection efficiency in serum-starved cells. This led to the establishment of two new serum-free cell lines: HEK-293T SF Luc2P/NF-kB and HEK-293T SF Receptor A, exhibiting stable viability and doubling times. When utilized in bioassays, these cell lines effectively mimicked the drug Mode of Action, confirming their potential for biological testing.