Conditions for maintenance of hepatocyte differentiation and function in 3D cultures
Niklas Handin,
Evgeniya Mickols,
Magnus Ölander,
Jakob Rudfeldt,
Kristin Blom,
Frida Nyberg,
Wojciech Senkowski,
Jozef Urdzik,
Varun Maturi,
Mårten Fryknäs,
Per Artursson
Affiliations
Niklas Handin
Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden
Evgeniya Mickols
Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden
Magnus Ölander
Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden
Jakob Rudfeldt
Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala, Sweden
Kristin Blom
Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala, Sweden
Frida Nyberg
Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala, Sweden
Wojciech Senkowski
Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala, Sweden; Biotech Research & Innovation Centre (BRIC) and Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen, 2200 Copenhagen N, Denmark
Jozef Urdzik
Department of Surgical Sciences, Uppsala University, Uppsala, Sweden
Varun Maturi
Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden
Mårten Fryknäs
Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala, Sweden
Per Artursson
Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden; Corresponding author
Summary: Spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different conditions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of an in vivo like phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymal transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function - a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research.
