Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development

Abstract

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Reanalysis of Trichloroethylene and Tetrachloroethylene Metabolism to Glutathione Conjugates Using Human, Rat, and Mouse Liver in Vitro Models to Improve Precision in Risk Characterization

“… 39 The MPCC platform has been shown to maintain the viability and metabolic activity of hepatocytes from rodents and humans for several weeks. 39 – 42 These improvements further highlight the potential for using MPCC to investigate xenobiotic metabolism, especially in cases where uncertainties are to be addressed through direct comparisons of species-specific metabolism.…”

Two Birds with One Stone: A Novel Dithiomaleimide-Based GalNAc-siRNA Conjugate Enabling Good siRNA Delivery and Traceability

“…The uptake of the siRNA-GalNAc conjugates was evaluated in freshly isolated primary mouse hepatocytes [37]. After cells were incubated with 10 nM siRNA, a comparable robust uptake was observed with standard GalNAc-L96 and our DTM-GalNAc (Figure 3A, part 3 in Supplementary Information).…”

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

“…CYP1A2 and CYP2A6 activities were measured by incubating the cultures with 5 µM 7-ethoxyresorufin or 50 µM coumarin (Sigma-Aldrich) for 3 h and 1 hour, respectively. The metabolites, resorufin and 7-hydroxycoumarin (7-HC), generated from 7-ethoxyresorufin and coumarin, respectively, were quantified via fluorescence detection (excitation/emission: 550/585 nm for resorufin, and 355/460 nm for 7-HC) on the Synergy H1 reader 70 .…”