Mickols, E.; Primpas, L.; Oredsson, S.; Karlgren, M.

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, thus facilitating fully chemically defined and animal serum-free 3D PHH cultures. Specifically, we combined a previously developed animal-free substitute cocktail (Rafnsdottir et al., 2023) with a normoglycemic (5.5 mM glucose and 0.58 ng/mL insulin) chemically defined culture medium (Handin et al., 2021). 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 animal serum-free culture conditions for primary human cell cultures, paving the way for more reliable and ethical in vitro studies.