Improved predictive models for pre-clinical drug toxicity studies
Navarro, Maria Dolores
Navarro-Zornoza, Maria Dolores
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Increasingly, drug-induced liver injury is one of the main reason for drugs to be withdrawn from the market even after passing toxicity studies in pre-clinical and clinical trials because of risks of toxicity and ineffective treatments. Human immortalised hepatocyte cell lines used in drug testing are widely available, inexpensive and easy to culture. However, these cell lines are commonly known to have poor predictive capabilities and improved in vitro hepatic models are required for predicting hepatotoxicity of large numbers of compounds in drug discovery. In this study, the primary goal was to develop an improved in vitro human hepatic model using a combination of the C3A human hepatic cell line and human umbilical vein endothelial cells (HUVECs), for prediction of acetaminophen (APAP) hepatotoxicity. Initial experiments showed that co-culture of HUVEC:C3A in EGM-2, an endothelial medium, was essential to support both cell types, and that co-cultures maintained the initial cell seeding ratio of 1:1 (HUVEC:C3A) after 3 days. Phenotyping of co-cultured cells using platelet endothelial cell adhesion molecule (PECAM-1/CD31) for HUVECs, and hepatic epithelial (EpCAM) markers for C3As demonstrated that at ratio 1:1 (HUVEC:C3A), there is cross-talk between HUVECs and C3As and cells in co-culture showed properties of self-organisation. This interaction resulted in improved hepatic metabolic activity in vitro in respect of albumin synthesis and cytochrome P450 activity. Treatment with low (5 mM), intermediate (10 mM) and high doses (20 mM) of APAP, showed that prediction of hepatotoxicity using specific kits for cell viability and mitochondria function, was significantly improved in C3As in the presence of HUVECs, thus demonstrating an in vitro human hepatic co-culture could be an invaluable model for drug toxicity studies. We observed that the intermediate APAP dose had no effect on cell viability and mitochondrial function in co-cultures, whilst by comparison both lactate levels and oxidative stress were perturbed in mono-cultures. Co-cultures also up-regulated expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in HUVECs following APAP exposure, which may be important in modulating the toxic effect of APAP on C3As. To further improve the in vitro liver-like model, Matrigel™ was incorporated to promote vascular formation by HUVECs and support hepatic organization, migration and function of C3As. In HUVEC mono-cultures, Matrigel™-promoted vascularization, haptotaxis and self-organization and in HUVEC:C3A co-cultures formation of structures reminiscent of liver sinusoids and maintenance of hepatic albumin synthesis and CYP3A4 activity. Time-lapse imaging showed haptotactic migration of hepatocytes towards endothelial cells, with Matrigel™ likely having a chemotactic effect on HUVECs and C3As, resulting in interconnected vascular network. APAP inhibited angiogenesis in HUVEC mono-cultures whereas APAP had no effect in HUVEC:C3A co-cultures. In conclusion, the development of an in vitro human organotypic co-culture model of HUVECs and C3As significantly enhanced hepatic function, demonstrated by significant improvement in hepatic metabolism, evidence of greater resistance to APAP toxicity, and improved cell-cell communication. Co-cultures markedly modulated APAP hepatotoxicity compared with C3A mono-cultures. Furthermore, co-culture of HUVECs and C3As using a complex basement membrane biomatrix (Matrigel™) produced a self-assembling interconnected vascular network, improved hepatocyte function as well as reproducibility of responses to APAP toxicity. The application of the described co-culture models may improve the accuracy, efficacy and predictive power of drug toxicity testing strategies in drug development.