Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

Abstract

Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to de-differentiating monocultures, co-culture with non-parenchymal cells maintains PHH functions for 2-4 weeks. However, since the functional lifespan of PHHs in vivo is 200-400 days, it is desirable to further prolong PHH functions in vitro towards modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned co-cultures (MPCC) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in non-starved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated adenosine monophosphate activated protein kinase and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating adenosine monophosphate activated protein kinase using metformin or growth-arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher druginduced cytochrome-P450 activities versus non-starved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime towards enabling clinically-relevant drug screening. S AMPK-adenosine monophosphate activated kinase, CYP-cytochrome P450, Cmax-The average maximal concentration the drug reaches in patient's blood after administration, GA-growth arrested, Met-Metformin, MPCC-Micropatterned co-culture, IC50-Drug concentration that causes a 50% reduction in assay endpoint.