Development And Preclinical Study Of A Human-derived Bioartificial Liver

Acute liver failure (ALF) is a devastating clinical syndrome of viral hepatitis, with the mortality rate more than 80% when treated by traditional supportive therapy. Liver transplantation is the clinically proven effective treatment of ALF. However, because of the shortage of donor liver, many patients die before an organ becomes available. Bioartificial liver support system (BALSS) is the most promising device to provide comprehensive liver functions support in vitro, so that ALF patients could be bridged to liver transplantation or liver regeneration. The critical biological material of BALSS is hepatocytes. Freshly isolated porcine hepatocytes and human liver tumor cells have been used as cell materials of BALSS, but because of the possibility of allergic reactions, viral infections and tumorigenesis, clinical applications of these types of BALSS were strictly limited. We established immortalized human hepatocytes (IHH) by transfections of primary human hepatocytes with simian virus 40 large T (SV40T) antigen and human telomerase reverse transcriptase (hTERT) genes. In the preliminary research, IHH showed high proliferative activity and could be cultured on microcarriers with high cell density. No tumorigenesis was found at the inoculation sites in the nude mice. The expressions of mRNAs and proteins involved in main functions of normal liver such as metabolism, detoxification and biosynthesis could be detected in IHH. After incubation the sera of ALF patients with IHH, the concentration of total bilirubin in the sera decreased, and the level of urea increased progressively, suggesting detoxification of bilirubin and ammonia by IHH.Objectives:To develop a human-derived bioartificial liver (hBAL) device using large-scale cultured IHH on microcarriers. To evaluate hBAL functions by perfusion detoxification of large volume of ALF plasma in vitro. To test the safety and efficacy of hBAL treatment in mice and rhesus monkeys with ALF. To provide evidences for hBAL to enter Phase I clinical trial.Methods:A stirred method was used to culture IHH on microcarriers on a large scale.ALF model was established by intraperitoneal injections of CCl4 in nude mice. The microcarriers cultured with IHH were intraperitoneally injected to ALF animal for dialysis, the ALF animals treated with empty microcarriers were used as control. The efficacy of the dialysis was evaluated by plasma aminotransferase, histopathological examinations and survival rate of ALF mice. The immunohistochemical staining for al-antitrypsin in microcarriers with IHH was performed to analyze their viability and function.The microcarriers cultured with IHH were loaded into a container to construct IHH-bioreactor, which connected to a peristaltic pump to perfuse against 400 mL ALF plasma for 6 h. After that, IHH-bioreactor was renewed for 18 h and again used for perfusion detoxification for 6 h, such perfusion-renew cycles were repeated for 6 times. The concentrations of plasma total bilirubin and urea were detected to evaluate the detoxification function of IHH-bioreactor in vitro.AlF model of Rhesus Macaque were induced by 2 intravenous injections of D-galactosamine (D-GalN) at time interval of 48 h. hBAL was developed by connecting IHH-bioreactor to blood purification device.2 rhesus monkeys with ALF were treated respectively by hBALs, either with continuous treatment modality of twice 5 h hBAL treatments performed successively or interval treatment modality of 5 h hBAL treatment and then 19 h interval, this treatment-interval cycles repeated for 3 times. Serum parameters of aminotransferase, bilirubin, blood ammonia, urea, bile acid, prothrombin time, prothrombin activity, fibrinogen and survival time and adverse reactions of ALF rhesus monkeys treated by hBAL were investigated to evaluate the efficacy and safety of hBAL. Chromosome karyotype and tumorigenicity in SCID mice of IHH was analysed to evaluate IHH safety.Results:Large-scale cultivations of IHH on microcarriers with high cell density were achieved with culture volume of 500-600mL. The plasma aminotransferase was increased over 10 times in ALF mice 24 hours after CCl4 administrations. All mice with ALF without any treatment or treated with empty microcarriers died within 48 hours and extensive hepatocellular necrosis was demonstrated by histopathological examinations. However, the survival rate of ALF mice treated with IHH-microcarriers was 83.3% within 48 hours and maintained to long-term survival. The histopathological examinations showed that hepatocytes necrosis was significantly reduced in ALF mice treated with IHH-microcarriers as compared with controls and immunohistochemical staining indicated that IHH on microcarriers was viable and expression of al-antitrypsin.The level of plasma total bilirubin decreased by 18% after 6 h of perfusion detoxification by IHH-bioreactor, which further decreased by 41% after 6 times of detoxification-renew cycles. Plasma urea level elevation began at the 4th detoxification-renew cycle and totally increased by 22% at the end of the 6th cycle. The level of total bilirubin and urea of the ALF plasma treated by empty microcarriers control did not changed significantly.ALF model of Rhesus Macaque were successfully induced by 2 intravenous injections of D-GalN at time interval of 48 h. Serum transaminase of rhesus monkeys increased 44 times, total bilirubin increased 5.4 times, blood ammonia increased 2.5 times compared those of normal rhesus monkeys. Prothrombin time increased to more than 75 seconds, and prothrombin activity decreased to less than 10%. The untreated ALF rhesus monkey had the clinical manifestations of bad-response, reduced intake of food and water, vomiting, oliguria, and die within 63 hours.Continuous modality of hBAL treatment for 10 hours administrated at 48 h after ALF induction could improve the blood biochemical indices of ALF rhesus monkey:ALT decreased by 70～80%, bilirubin decreased by about 70%, the urea level increased so that ammonia did not continue to rise, total bile acid decreased by 75%. These indices rebounded after the termination of hBAL treatment, with the peak on 4th day after induction of ALF. Coagulation parameters could not be improved by hBAL treatment. The total survival time was 7.6 days. Histopathologic examination of the liver tissue demonstrated alleviation of hepatocellular necrosis and hepatic congestion compared with livers of untreated ALF rhesus monkeys.Interval modality of hBAL treatment administrated at 24 h after ALF induction could decrease aminotransferase level by about 50% with each treatment of 5 hours, which gradually increased during the intervals of treatments. However, the rebounded peak on 4th day after induction of ALF was much lower than that of the continuous modality of hBAL treatment, only 39% of that. Interval hBAL treatment could inhibit the bilirubin increase, with the rebounded peak only 12% of that of continuous treatment. Urea levels after interval hBAL treatments were significantly higher, about twice than that of the continuous treatment, in the same time, blood ammonia maintained normal levels. Interval hBAL treatment also inhibited the increase of TBA and improved coagulation parameters, which returned to normal levels on 7th day after induction of ALF. ALF rhesus monkey was cured and maintained long-term survival.The hBAL device had stable operation performance, with all the parameters maintained within the normal range. Adverse reactions of hypotension and low blood calcium were observed and could be relieved after symptomatic treatments.Karyotype analysis showed a diploid pattern of IHH. No tumorigenesis were observed after IHH were inoculated subcutaneously and in the liver of SCID mice for 6 months.Conclusions:IHH cultured on microcarriers could be employed as a cell source for development of a renewable human-derived bioartificial liver. hBAL had detoxification function in vitro, and could reduce the bilirubin level and increase urea level of 400 mL ALF plasma. hBAL treatments could significantly prolong survival time and improve blood parameters of ALF mice and ALF rhesus monkeys. hBAL treatments demonstrated satisfactory clinical efficacy and safety. The interval hBAL treatment modality was more effective than the continuous treatment modality. Earlier hBAL treatments demonstrated better prognosis of ALF rhesus monkey.