Examination of common culture medium for human hepatocytes and engineered heart tissue: Towards an evaluation of cardiotoxicity associated with hepatic drug metabolism in vitro

Abstract

Cardiotoxicity associated with hepatic metabolism and drug-drug interactions is a serious concern. Predicting drug toxicity using animals remains challenging due to species and ethical concerns, necessitating the need to develop alternative approaches. Drug cardiotoxicity associated with hepatic metabolism cannot be detected using a cardiomyocyte-only evaluation system. Therefore, we aimed to establish a system for evaluating cardiotoxicity via hepatic metabolism by co-culturing cryopreserved human hepatocytes (cryoheps) and human iPS cell-derived engineered heart tissues (hiPSC-EHTs) using a stirrer-based microphysiological system. We investigated candidate media to identify a medium that can be used commonly for hepatocytes and cardiomyocytes. We found that the contraction length was significantly greater in the HM Dex (-) medium, the medium used for cryohep culture without dexamethasone, than that in the EHT medium used for hiPSC-EHT culture. Additionally, the beating rate, contraction length, contraction speed, and relaxation speed of hiPSC-EHT cultured in the HM Dex (-) medium were stable throughout the culture period. Among the major CYPs, the expression of CYP3A4 alone was low in cryoheps cultured in the HM Dex (-) medium. However, improved oxygenation using the InnoCell plate increased CYP3A4 expression to levels comparable to those found in the human liver. In addition, CYP3A4 activity was also increased by the improved oxygenation. Furthermore, expression levels of hepatic function-related gene and nuclear receptors in cryoheps cultured in HM Dex (-) medium were comparable to those in the human liver. These results suggest that the HM Dex (-) medium can be applied to co-culture and may allow the evaluation of cardiotoxicity via hepatic metabolism. Moreover, CYP induction by typical inducers was confirmed in cryoheps cultured in the HM Dex (-) medium, suggesting that drug-drug interactions could also be evaluated using this medium. Our findings may facilitate the evaluation of cardiotoxicity via hepatic metabolism, potentially reducing animal testing, lowering costs, and expediting drug development.

Fig 1. Contractile properties of human iPS cell-derived engineered heart tissues (hiPSC-EHTs).

hiPSC-EHTs were cultured in EHT medium for contractile property analysis and then cultured in the HM or HM Dex (-) media. Thereafter, the contractile properties of hiPSC-EHTs were analyzed after 0.5, 24, 48, and 72 h of culture. Movie images were analyzed using SI8000 software. The graph bar shows the average mean, and the error bar shows the standard error (n = 4). * Statistically significant change compared to the 0.5 h values (p < 0.05).

Fig 2. Heatmap showing cardiomyocyte-specific gene expression in human iPS cells-derived engineered heart tissues (hiPSC-EHTs).

hiPSC-EHTs were cultured in EHT medium for over 1 week and then in EHT medium, HM medium, or HM Dex (-) medium for 72 h. RNA was collected from the tissues at the endpoint and analyzed using qPCR (n = 4). The color of the Heatmap shows the value of log2 (relative expression level compared to the human left ventricle).

Fig 3. Cytochrome P450 expression in cryopreserved human hepatocytes (cryoheps).

Cryoheps were cultured in EHT medium, HM medium, or HM Dex (-) medium for 72 h from the day of seeding. Polystyrene (PS) plates and InnoCell plates were used for the culture. RNA was collected from the cells at the endpoint and analyzed by qPCR. The graph bar shows the mean of the relative expression levels compared to the human liver, and the error bar shows the standard error (n = 3). The green, red, and blue lines show the mean, maximum, and minimum values of expression in the 22 lots of cryoheps under vendor-recommended conditions [34]. * Statistically significant increase in the InnoCell plate compared with the values in the PS plates (p < 0.01).

Fig 4. Expression of genes related to liver function in cryopreserved human hepatocytes (cryoheps).

Cryoheps were cultured in the EHT medium, HM medium, or HM Dex (-) medium for 72 h from the day of seeding. PS plates and InnoCell plates were used for the culture. RNA was collected from the cells at the endpoint and analyzed by qPCR. The graph bar shows the average mean of the relative expression levels compared with the human liver, and the error bar shows the standard error (n = 3).

Fig 5. Expression of nuclear receptors in human cryopreserved hepatocytes (cryoheps).

Cryoheps were cultured in the EHT medium, HM medium, or HM Dex (-) medium for 72 h from the day of seeding. Polystyrene plates and InnoCell plates were used for the culture. RNA was collected from the cells at the endpoint and analyzed using qPCR. The graph bar shows the average mean of the relative expression levels compared with the human liver, and the error bar shows the standard error (n = 3).

Fig 6. Induction of cytochrome P450 expression in human cryopreserved hepatocytes (cryoheps).

Cryoheps were cultured in the HM or HM Dex (-) media with inducer for 48 h from day 2 after seeding. Polystyrene and InnoCell plates were used for the culture. RNA was collected from the cells at the endpoint and analyzed by qPCR. The graph bar shows the average mean gene expression fold change upon exposure to the inducer (OM: Omeprazole, RIF: Rifampicin, PB: Phenobarbital), and the error bar shows standard error (n = 3).

Fig 7. Cytochrome P450 (CYP) activity in human cryopreserved hepatocytes (cryoheps).

Cryoheps were cultured in the HM Dex (-) medium on polystyrene (PS) or InnoCell plates for 72 h from the day of seeding. PS and InnoCell plates were used for the culture. Cryoheps were incubated in the HM medium Dex (-) containing a cocktail of CYP probe substrates. After 60 min of incubation, the incubation media were collected and the metabolites were measured using LC-MS/MS. The graph bar shows the average mean CYP activity and the error bar shows the standard error (n = 4). * Statistically significant increase in the InnoCell plate compared to the values in the PS plates (p < 0.01).