| Backgroud:Acute liver failure is severe illness, the progress is rapid and the prognosis is poor.. How to improve survival rate of patients with acute liver failure are urgent need to solve.Despite medicine has made great progress in the comprehensive treatment in recent years, the mortality rate is still high. Liver transplantation can significantly reduce mortality of patients with liver failure, but because of severe shortage of donor liver,, in fact, less than 1/3 of the patients received liver transplantion, the majority of patients died in the process.of awaiting for liver. Based on this premise, domestic and foreign scholars designed different types of artificial liver support system and hope that use a mechanical, physical and chemical or biological device to clear the accumulation in patients with a variety of harmful substances to supplement the required material in vitro, to improve the the environment of patients, and to replace some functions of liver. Creating conditions for liver regeneration and recovery, or waiting for liver to transplantation, thereby reducing the death rate in patients with liver failure.After more than 50 years of development, the technology of artificial liver support system has gradually development At abroad,most scholars, according to the composition and nature of the artificial liver.ALSS is divided into the following three categories:(1) non-bioartificial liver (2) bioartificial liver (bioartificial liver, BAL) (3) hybrid bioartificial liver (hybrid bioartificial liver, HAL).. HAL represents the direction of development of ALSS.Whether BAL or HAL, liver cells are the core, the function of BAL or HAL almost entirely dependent on liver cells functions used in the bioreactor.there are some categories cell can be used as seed cell for BAL or HBAL,but there is no one can fully meet the needs of clinical. For example, as the source of human liver cells are limited, the ethical aspects of fetal liver cells, liver tumor cell line-derived and other reasons, procine liver cells, a potential xenograft cellular immune response, species differences of protein and widespread in pigs endogenous retrovirus (procine endogenous retroviral, PERV). Our preliminary study found that Chinese human liver cell line(CL-1)is high differentiation and biological metabolic functions well, and the CL-1 cells derived from normal liver tissue, compared with other liver from tumor-derived cell lines is more safety, but it is not yet that use the CL-1 cells as seed cells for BAL or the HAL.So far four kinds of BAL or the HBAL system has been startedⅡ-Ⅲa clinical validation, most of which use hollow fiber bioreactor, Oxygen dissol ved in plasma, with the plasma flow to supply oxygen and nutrients liver cells Preliminary study proved that hollow fiber bioreactor, is safe and effective, no significant adverse events occurred. However, due to lack of oxygen carrying capacity of blood plasma and, therefore, these systems are far from the oxygen supply can not meet the needs of liver cells in bioreactor,, the majority of liver cells may be in a hypoxic state, which led to reduced activity of liver cells and liver cell dysfunction, eventually leading to reduced efficacy of these artificial liver support system. And hollow fiber bioreactor in the uneven distribution of liver cells, poor cell adhesion, membrane pollution and congestion, cell growth or excessive gas production would undermine the fiber, not only affect cell function,.but also affect observation cell function in bioreactor.To address problems of bioreactor supply oxygen for liver cells, We are in co-operation with the Chinese Academy of Sciences has developed a perfusion bioreactor. The reactor is made of transparent organic plastic with two imports and two exports and this reactor take round-trip time to do a 180-degree rotation when in the treatment, liver cells in direct contact with plasma in the bio-reactor, liver cells can be full metabolism and bio-transformation function, and the biological part of the HAL system, has an independent of the device to oxygen, oxygen into the serum through the membrane oxygenator, the supply of reactor in the liver cells to ensure that bio-reactor in the liver cells, adequate oxygen supply.In this study, we use CL-1 cell co-cultured with microcarrier (cytodex 3). In the fifth day, put the cells into reactor, combined with the plasma perfusion devices, biological reaction tank, peristaltic pump, membrane oxygenator, etc. to build a simple, novel HAL,. Specific research contents include the following three parts:1:Configuration of a combination can reflect some changes in the surem of patients with liver failure, and can be stable stored at 37.5℃;2:Construction hybrid bioartificial liver and evaluation its function:in vitro;3:Study.the safety of CL-1 cell as seed cells for the HALPart I Configuration of a combination can reflect some changes in the surem of patients with liver failureObject To provide a composition, on the basis of the serum component changed in patients with hepatic failure, for detecting the function of bioartificial liver in vitro and its preparation.Method (1)The major components of the composition:non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride are dissolved in cell medium:DMEM and make the composition contains 10%Serum of Fetal bovine, the level of non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride was 171-342μmol/L,80-160μmol/L,20-40μmol/L and 240-40μmol/L, respectively. (2)We convert non-conjugated bilirubin into non-conjugated bilirubin disodium, then, follow the concentration of above, non-conjugated bilirubin disodium, chenodeoxycholic acid, bile acid and ammonium chloride dissolved in serum-free cell culture medium. Then,at37℃, we detect the concentration of various substances of composition at 0,24 and 48-hour, respectively.Result The composition is stable at 37.5℃and the concentration of various substances of the composition were no significant difference, at 0,24 and 48-hour(p>0.05).Conclusion At 37.5℃, within the scope of the concentration:171-342μmol/L of non-conjugated bilirubin,80-160μmol/L of chenodeoxycholic acid,20-40μmol/L of cholic acid and 240-400μmol/L of ammonium chloride, the composition is stable. We could use it to test the function of bioartificial liver in vitro in baseline.PartⅡConstruct a new hybrid bioartificial liver and evaluate its function:in vitroobjectiveThis study aimed to design a new type of hybrid bioartificial liver and evaluated its efficacy through purify the simulation serum of liver failure, to explore the feasibility of its clinical applicationMethod CL-1 cells and microcarrier are co-culture in three-dimensional with microgravity,in the fifth day, CL-1 cells and microcarrier are poured into in the bioreactor in sterile environment, the new hybrid BAL system construction: bio-reactor plus hemoperfusion device,the volume of bio-reactor is about 300 ml (cell volume 1.0×109 cell density 4.0×108/ml), oxygen and carbon dioxide are intermittent injected in bio-reactor of, the time ratio is 110:10 (ie, in 2 minutes, inject oxygen 110s, inject carbon dioxide 10s). Simulated liver failure serum is 20ml/min, and 6ml/min of them are separated through the plasma separator and inflow into the blood perfusion device, after purified by perfusion device,4ml/min inflow into the bioreactor, while bio-reactor parts,100ml/min from the loop, and through the second sub-paste to 4ml/min flow from the bioreactor, the bio-reactor maintain at 37.5℃. Detection the compositions of simulated liver failure serum before the cycles,1,12, 24,48,72,96 and 120 h of cycles,such as unconjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride concentrations,we also measured AL T, AST, LDH level in the bio-reactor. MTT way to determine the rate of liver cells alive.ResultThere was no liquid leaks from the pipeline and no liver cell and micro-carrier leak from the reactor throughout the course of treatment; the total number of liver cells and the viability was no significant change within 24 h, the new hybrid BAL system has a strong ability to clear toxins in patients with liver failure and lidocaine metabolism.ConclusionsWe therefore conclude that (1)In the newly designed HAL system, CL-1 cells can keep their viability and function in vitro, and (2)the HAL appears to be effective in purified the simulation serum of liver failure, with improvement non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride. The safety and efficacy of this HAL system seems to be promising for animal experimental.PartⅢStudy the safety of CL-1 cells and its debris tumorigenicityObjectiveEvaluate the safety of CL-1 cells as the seed cell for HALMethodCulture the CL-1 cells to the fifth dayuse the 2.5g/L trypsin digestion and centrifugation, DMEM transferred cell density to 1.0×1010/L, under the-70℃, CL-1 cells repeated freezing and melting three times, so that cell lysised,0.2ml cell lysis solution were inoculated to the five nude mice,the neck and the back (n= 10)). In control group,0.2ml CL-1 cells solution with the same density s were inoculated into the the other five nude mice (n= 10),typan blue exclusion rate of living cells, observation subcutaneous tumorigenicity.4 weeks after, the tumor tissues were cut from the injection site and hematoxylin-eosin (HE) staine. liver, lungs and brain tissue of nude mouse.Result:After inoculation 4 weeks, there was no cultivation of tumor in the inoculation site in the experimental group; in the control group,10 inoculation sites appear tumors, tumorigenic rate was 100%.Cut the cultivation of subcutaneous tumors in nude mice, gross specimen shows:tumor was smooth, the ball type, clear boundaries, there is a complete capsule. There was no tumor founed in Liver, lung and brain tissue.ConclusionCL-1 cell line could be safety as seed cell for HAL...
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