Animal product-free formation and cultivation of three-dimensional primary hepatocyte spheroids

Three-dimensional (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 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 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 with a normoglycemic (5.5 mM glucose and 0.58 ng/mL insulin) chemically defined culture medium. Morphological and viability evaluations, along with global proteomics data, demonstrated that serum-free cultured 3D PHH have comparable viability and functional performance of cytochrome P450s, rendering this medium useful for long-term studies and in vitro absorption, distribution, metabolism, excretion, and toxicity 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. SIGNIFICANCE STATEMENT: Most in vitro cell models rely on FBS. However, the use of FBS leads to inconsistent experimental results and raises serious ethical concerns. In this study, a chemically defined animal product-free cell culture medium with physiologically relevant levels of key hormones and nutrients for liver spheroid cultures was developed and evaluated. This study marks a significant advancement in the development of animal serum-free culture conditions for primary human cell cultures used in drug disposition studies.