Development Of Micro Hollow Fiber Reactor For Research On Drug Hepatotoxicity

With the rapid development in discovery of new drugs, highly effective in vitro models were needed to evaluate drug hepatotoxicity. However, traditional animal models in vivo were not well adapted for the high-throughput screening in drug discovery, due to the shortcomings of time consuming, operation complexity and high cost. Recently, various drug metabolic models in vitro were invented. In routine drug research, primary hepatocytes cultured in 96-well plates were used for the high-throughput screening in vitro. However, hepatocytes cultured in 96-well plates lost their liver-specific functions and cytochrome P450 activity rapidly. The drawbacks of monolayer culture limited its application in evaluating the hepatotoxicity of long-term drug exposure. Therefore, long-term models in vitro were suggested for research on drug hepatotoxicity instead of monolayer culture.This thesis was divided into five sections. Section 1 reviewed drug discovery on hepatotoxicity and hepatocytes culture in vitro. At the end of this section, research outline was illustrated for establishing a micro-bioreactor for 3-D cells culture in drug hepatotoxicity research.In Section 2, a series of experiments were conducted to explore the appropriate materials for the micro-bioreactor. The micro hollow fiber reactor (MHFR) was composed of several PS hollow fibers with 100kDa molecular weight cutoffs, a piece of silicone tubing and two silicone plugs. Freshly isolated rat hepatocytes were mixed with type I collagen solutions (obtained from rat tail) and then entrapped within hollow fibers. The entrapped hepatocytes maintained cell viability and liver-specific functions for 14 days.In section 3, we focused on the feasibility of MHFR in drug hepatotoxicity research. We found that the hepatocytes entrapped in MHFR were more sensitive to acetaminophen (2.5-20 mmol/L), which reflect the hepatotoxicity invivo well. This could be due to the better maintainance of CYP450 activity and simulate the in vivo micro-envelopment properly. Then, low concentrations of isoniazid (15 mg/L) and rifampicin (10 mg/L) were adopted to describe the feasibility of MHFR in the research of drug hepatotoxicity under low doses. Drug hepatotoxicities were apparent after 3 days' treatment, while no significant hepatotoxicities were detected in monolayer culture for 5 days' treatment.In section 4, we discussed the protection effect of MHFR against drug hepatotoxicity. Acetaminophen acted as a hepatotoxicity drug, and glutathione as a protective drug. The results showed that protective effect of GSH against acetaminophen toxicity decreased after 24 hours' treatment in hepatocytes monolayer culture and only reduced after 5 days' treatment for those entrapped within MHFRs. It implied that the MHFR, which longer maintained the enzyme activity involved in GSH protection, was more effective than monolayer culture.Finally, this paper was summarized in section 5. And the application of MHFR in drug metabolism, Chinese medicine discovery and drug clinical forecast with human hepatocytes instead were prospected in the end.