Long‐term maintenance of functional primary human hepatocytes using small molecules

Abstract

The immediate deterioration of primary human hepatocytes (PHHs) during culture limits their utility in drug discovery studies. Here, we report that a cocktail of four small molecule signaling inhibitors, termed YPAC, is useful for maintaining various hepatic functions of PHHs, including albumin and urea productivity, glycogen storage, and cytochrome P450 (CYP) expression. Most importantly, we found that YPAC allows PHHs to retain enzymatic activities of CYP1A2, CYP2B6, and CYP3A4 even after 40 days of culture, and that inducibility of CYP3A4 activity in response to the prototypical inducers rifampicin and phenobarbital is also maintained. Our novel approach could facilitate drug discovery studies.

Chemical‐based primary human hepatocyte monolayer culture for the study of drug metabolism and hepatotoxicity: Comparison with the spheroid model

“…Expression levels are shown relative to the average of all samples (pink and green for higher or lower than average, respectively). Genes were selected based on the study by Bell et al 17 and Katsuda et al 20 The culture duration of each sample is presented by the colors on the bottom of each column (for the color code, see Figure 2C). B, Principal component analysis (PCA) plot of core ADME and EMT genes based on Figure 2A; the shape of symbols (score plot) indicates the sample condition and the color of symbols shows the in vitro culture period (for the keys, see Figure 2C).…”

“…Recently, two groups managed to maintain PHH monolayers with regular hepatocyte phenotype and functional activities for more than 1 month on collagen‐I‐coated plates by adding cocktails of small‐molecule signaling inhibitors in formula‐defined media 20,21 . However, it has not yet been verified whether such chemical‐based long‐term monolayer PHH models are suitable for drug ADME and toxicology applications, and how they compare in this respect with the abovementioned tissue engineering models.…”

Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development—Current State-of-the-Art and Future Perspectives

“…Further mechanistic insights were provided by two recent studies that presented small-molecule combinations for the partial inhibition of dedifferentiation in 2D culture. One strategy used an adenylate cyclase activator combined with inhibitors of Notch, TGFb, and BMP signaling and a blocker of Wnt secretion (Xiang et al, 2019), whereas the other strategy combined TGFb inhibition with inhibitors or rho-associated protein kinase and mitogen-activated protein kinase as well as an activator of Wnt/b-catenin signaling (Katsuda et al, 2020). Notably, although both approaches report strongly improved viability and hepatic functions for up to 40 days in monolayer culture, substantial alterations of gene expression levels compared with freshly isolated hepatocytes remain.…”

Tissue-Engineered Bile Ducts for Disease Modeling and Therapy

“…35 Some efforts have been made to the in vitro expansion of PHHs, which retain their hepatic features. [36][37][38][39] Although the PHHs were dedifferentiated to a biphenotypic state by defined media conditions, the biliary differentiation has not been studied in-depth for this cell source.…”