Animal product-free culture conditions for 3D primary human hepatocyte spheroids improve long-term viability and drug metabolism

3D cultures of primary human hepatocytes (3D PHH) are successfully used to reduce and replace the use of animal experiments in biomedical research. Yet, the initial formation of 3D PHH is highly dependent on the supplementation with fetal bovine serum (FBS). However, the molecular composition of FBS and its effects on cultured cells are poorly understood. Moreover, FBS is prone to batch-to-batch variation, immunogenic risk, and lack of adherence to the replacement, refinement and reduction (3Rs) of animal experiments. Here, we demonstrate that FBS can be fully replaced by animal-free substitutes. Specifically, we combined a previously developed animal-free substitute cocktail with a normoglycemic chemically defined culture medium. Morphological and viability evaluations along with global proteomics data, demonstrated that serum-free cultured 3D PHH have equal or superior viability and functional performance of cytochrome P450s, rendering this medium useful for long-term studies and in vitro ADMET applications. This study marks a significant advancement in the development of serum-free culture conditions for primary human hepatocytes, paving the way for more reliable and ethical in vitro studies.