| Background:Liver failure is the inability of the liver to perform its normal detoxification, biosynthesis, and/or biotransformation functions. The clinical presentation of liver failure includes a prolonged prothrombin time, encephalopathy, and jaundice. Regardless of the etiology, liver failure can be divided into two categories:acute liver failure (ALF) or acute on chronic liver failure (AoCLF). Both are accompanied by high mortality. Liver transplantation is still the only ultimate solution for end stage liver failure, but its application is hampered by a world-wide scarcity of donor organs. In this context, extracorporeal artificial liver support systems have been expected to be a bridge to transplantation or to provide an opportunity for the native liver to regenerate.Depending on whether they are loaded with metabolically active hepatocytes or not, these systems can be roughly classified into two types:non-biological liver (NBL) or bioartificial liver (BAL). NBLs have proven to be useful for improving biochemical parameters and clinical symptoms in many cases, but the prognostic benefits have not yet been fully reflected. It is widely accepted that a NBL, which can only detoxify, is insufficient to support liver failure patients, while in theory an ideal hepatocyte-filled BAL based on hepatocyte-filled bioreactor could provide most or even all normal liver functions. However, until now, only two randomized controlled clinical trials exploring the effectiveness of BALs have been reported, and the results were not encouraging. Probably, BALs alone can not be competent for the job at the present level of technology. Future extracorporeal artificial liver support systems should be the combination of cell-filled bioreactors and NBL components.To develop such an extracorporeal artificial liver support system, three things are essential:1) an appropriate cell source;2) a bioreactor capable of providing in vivo-like environments for cells; and3) an extracorporeal circulation device which can control not only the cell-filled bioreactor, but also most types of existing NBL components.We recently designed a choanoid fluidized bed bioreactor filled with alginate-chitosan (AC) encapsulated primary porcine hepatocytes and proved it could prolong the survival of pigs with acute liver failure (ALF) induced by D-galactosamine injection. The potential risk of zoonotic transmission, however, might exist, which limits its clinical application. Therefore, developing a safe cell source for BAL is necessary. For several years, we have been dedicated to establishing immortalized hepatocyte line. Although these cell types have unlimited expansion capabilities in vitro, continuous expression of simian virus40large T antigen (SV40LT) might be tumorigenic. To solve this problem, we established a reversibly immortalized cell line (HepLi-4) by transfection of primary human hepatocytes with drug-medicated Cre/LoxP recombination. By doing so, the immortalizing Oncogene (SV40LT) can be excised programly. In order to demonstrate the clinical potential of HepLi-4cells in BAL, we carried out experiments on large animal models.Another problem facing us is all extracorporeal circulation device applied in our research were not domestic products. For example, the extracorporeal circulation device used to evaluate our choanoid fluidized bed bioreactor and HepLi-4cells is Plasauto-iQ (Asahi Medical, Japan). Also, functions of these commercial devices are rather limited. This is our motivation to develop a novel extracorporeal circulation device which can control not only most of the existing modes of NBL, such as plasma exchange (PE), hemodialysis (HD) and plasma adsorption (PA),but also BAL treatment modes. Also, all treatment modes can be applied either jointly or sequentially. To build the prototype of such a device is the first step. Part I Evaluation of a bioartificial liver system loaded with reversibly immortalized human hepatocytes in pigsObjective:To evaluate the bioartificial (BAL) system loaded with our newly established reversibly immortalized cell line (HepLi-4) in Chinese experiment miniature pigs with acute liver failure (ALF).Methods:HepLi-4cells expressing Tamoxifen-dependent Cre recombinase were expanded in roller bottles and SV40LT genes were removed by keeping the cells in culture media containing500nM4-hydroxytamoxifen for5-7days. Alginate-chitosan (AC) microbeads containing HepLi-4cells were produced via single-stage procedure. ALF was induced in Chinese experiment miniature pigs with intravenous injection of D-galactosamine at a dose of1.5g/kg body weight. Fifteen ALF pigs were allocated to three groups:a BAL group, receiving BAL treatment with encapsulated HepLi-4cells (n=5); a sham BAL group (device control), receiving cell-free BAL treatment (n=5); a ALF group (baseline control), receiving intensive care only (n=5). Survival time and biochemical parameters of pigs were measured. Before and after BAL, microbead integrity and cell viability were tested, also, expression of liver-specific genes in HepLi-4cells was analyzed, adult human liver acted as a reference.Results:In BAL group, Fischer index was higher and serum indirect bilirubin level was lower compared with two control groups. Survival time in BAL group is longer than that in two control groups, but the difference is not statistically significant. After BAL, microbead integrity and cell viability did not decrease significantly. Gene expression analysis showed that the transcript levels of liver-specific genes in HepLi-4were retained after BAL, but significant variations were observed between HepLi-4and adult human liver.Conclusion:HepLi-4showed beneficial metabolic effects on ALF pigs in BAL, but is still not an appropriate cell source for BAL. More insights into interpreting the conditions for hepatocyte differentiation are needed. Part â…¡ Development of an extracorporeal circulation device of artificial liver systemObjective:To develop the prototype of an extracorporeal circulation device of artificial liver system which can control most of the existing modes of non-biological liver (NBL) and bioarticial liver (BAL) support treatment.Methods:The prototype of the extracorporeal circulation device consisted of a nonbiological section and a biological section. The control center was composed of an industrial personal computer, two serial communication ports and a peripheral component interconnect (PCI) digital input and output (DIO) card. Most components are integrated via the RS485buses. The software provided users an operation guide and a human-machine communication interface. Based on hardware and software design, real time status monitoring and regulation could be realized. Also, all parameter values during the treatment can be recorded for post hoc analysis. To verify our design, we tested the prototype of extracorporeal circulation device both in vitro and on miniature pigs.Results:The hardware and software of the prototype of the extracorporeal circulation device runned normally in all treatment modes. Functions of all components were verified. Also, timely detection of abnormal conditions in the clinical treatment could be fulfilled.Conclusion:The prototype of the extracorporeal circulation device has a good safety and user-friendly design.It can provide a reliable research platform for commercialization of the device. Also, it can act as a simulation training platform for relevant technical staff.
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