| Background:Liver is one of the most important organs in the body, which has a complex structure and a variety of physiological functions. Its main functions include oxidative detoxification, storing glycogen, metabolism of cholesterol, synthesis and secretion of proteins and some hormones, and secretion bile[1].Liver failure is a severe clinical syndrome and the mortality ranges from 60 to 90%.At present, there are three methods to treat liver failure, which are drug treatment, bioartificial liver (BAL) therapy and orthotopic liver transplantation (OLT) [3]. Orthotopic liver transplantation (OLT) is the most effective method by far[4]. But it is beacause the lack of donor, critical conditions and no enough time to wait for the suitable liver that the case fatality rate is high and prognosis is poor. The emergence of the bioartificial liver treatment is expected to be a transitional treatment for liver transplant operation..The basis of bioartificial liver treatment is the culture of liver cells in vitro. The bioreactor is the key part in BAL and itsfunction can directly affect the effect of BAL. The ideal bioreactor should satisfy the following conditions:1. provision of a good environment for liver cells to grow and metabolize; 2.adequate bidirectional transport of mass between media and liver cells; 3.protecting the liver cells from the damage of the host immune system;4. culturing enough cells to meet the needs of medical treatment; 5. minimization of the invalid space in the device. How to design the best new bioreactor to realize the large-scale cultivation of functional liver cells in vitro and meet the needs of clinical therapy is still the core problem in the development of BAL. After more than 20 years of development, the structures of bioreactor can be divided to five kinds:1. Flat plate and monolayer;2.hollow fiber;3. perfused beds or scaffolds;4. beds with encapsulated or suspended cells;5.other complicated configurations with compound bioreactors [8-12]. However, the existing bioreactors are far from ideal in terms of material biocompatibility,density and scale of cell culture(The ideal quantity of aritificial hepatocytes is about 1/5 to 1/10 of normal hepatocytes, namely 1-2 x 1010), and mass exchange between hepatocytes and plasma. The RCCS(Rotary Cell Culture System), designed by the National Aeronautics and Space Administration (NASA), has been widely applied in fields of tissue and cell culture. It adjusts the rotation speed of a full of culture fluid container to offset its inner cell sedimentation rate (ESR), enabling the simulation of three-dimensional microgravity environment. In this system, the cells in a state of weightlessness can firmly adhere on microcarriers, so it can promote the cell aggregation and three-dimensional growth. Meanwhile, it has many characteristics, such as a low shear,low turbulence and sufficient oxygen and nutrient exchange. This suspension culture technique provide good culture environment for the growth and metabolism of a variety of cells. It can realize high-density tissue culture, and maintain the differentiation specificity of the cultured cells. It is widely applied to the study of cell culture, for example fat stem cells, cartilage cells, liver cells and other cell culture. Zhang Yupeng cultured rat primary hepatocytes, using rotary cell culture system to simulate microgravity. In the simulated microgravity culture environment liver cells attached to microcarriers emerged three-dimensional structure and growed into characteristic liver cells and poly spheres of microcarriers. YaoXinyu cultured liver stem cells in three-dimensional and simulated microgravity conditions.It is proved that simulated microgravity environment can promote the growth of liver stem cells and maintain cell activity and phenotype. We expected to study the feasibility of microgravity bioreactor as a new generation of bio-artificial liver bioreactor. We imported the 500ml rotating and perfusion microgravity bioreactor (Rotary Culture MWTM RCMW) from the National Aeronautics and Space Administration (NASA) in America. But repeated experiments revealed that the RCMW bioreactor has serious flaws in the design:1. Horizontal force can not be balanced; 2.The efficience of Material exchange is low; 3.The supply and exchange of gas in the system are not effective. Our early experiment developed the fist generation of bi-directional rotation and perfusion bioreactor system by optimizing of the inner core and circular model of the RCMW bioreactor.The new system can provide a good environment for growth and metabolism of hepatocytes and increase the culture scale to meet therapy needs. It basically meet the requirements of the ideal bioreactor and provide a new direction for the study of bioreactor. But there are some inadequacies, mainly consisting of the following four questions:1. Imbalance of the pressure in horizontal direction; 2 The problem of circulation mode; 3.The F6 dialysis column of the System produces too much gas, which affects the operation of the system;4.Complex pipeline design is not convenient to operate. By optimizing the flaws of the first generation.of bi-directional rotation and perfusion microgravity bioreactor., we has successfully developed the second generation of bi-directional rotation and perfusion bioreactor system. This experiment shows us a new direction for the research of the artificial liver bioreactor.Objectives:To developed the second generation of bi-directional rotation and perfusion bioreactor system by optimizing the serious flaws in the first generation.of bi-directional rotation and perfusion microgravity bioreactor.Methods:by the power system and gas supply system, on the basis of the the first generation.of bi-directional rotation and perfusion microgravity bioreactor optimized building formed by the cell culture tank, the culture liquid pool, the power control system (peristaltic pump, the commutation groove), the composition of the first gas. supply system the second generation of the new two-way rotation perfusion microgravity bioreactor system. The the first generation.of bi-directional rotation and perfusion microgravity bioreactor, the second generation of bi-directional rotation perfusion microgravity biological reactor continued to microcarrier three-dimensional cultured for 7 days, and by the MTT assay, cell count, culture supernatants ALT, AST, albumin, urea concentration index measures were compared liver cell viability, quantity and functional differences. Statistical Methods:The measurement data were expressed as mean ± standard deviation (x ± s) said, using the SPSS 17.0 statistical software analysis. Data between the two groups mean compared using t-test analysis, a significant test of the level of a= 0.05.Results:The the irst generation.of bi-directional rotation and perfusion microgravity bioreactor designed by NASA has many disadvantages, including microcarrier accumulation, low exchange efficiency and dead culture space. The efficiency of mass transfer in t he first generation of of the bi-directional rotation and perfusion microgravity biore actor system is inceased. But there are four major problems:1. imbalance of the pre ssure in the system; 2. pump location; 3 the production of too much gas by the dialy sis column of the oxygenator 4. complex pipeline design. The second generation of bi-directional rotation and perfusion microgravity bioreactor system adequately add ress above issues. The inverted microscope and the MTT staining results show that t he quantity and viability of the human hepatocyte (C3A) in bidirectional bioreactor group are much better than the the first generation.of bi-directional rotation and perfusion bioreactor group. The growth curve results show that the density of the human hepat ocyte (C3A) was firstly increased and then decreased in both groups, and the peak o f the curve appears in day5. The density of human liver cells in the second generati on of bi-directional rotation and perfusion microgravity bioreactor group was signif icantly higher than the the first generation.of bi-directional rotation and perfusion bioreactor group from day1 to day 7 (P<0.05).The functional results show that the albumin an d urea concentration,which reach the peak on day5, also go up firstly and then grad ually go down in both teams. And the albumin and urea concentration in bidirection al bioreactor group is significantly higher than the first generation.of bi-directional rotation and perfusion microgravity bioreactor group from dayl to day7 (P<0.01). Besides, the concentration of ALT and AST in bidirectional bioreactor group is sign ificantly lower than RCMW group from dayl to day7 (P<0.05).Conclusion:This study successfully designed the second generation of bi-directional rotating and perfusion microgravity bioreactor by optimizing the existing problems in the the first generation.of bi-directional rotation and perfusion microgravity bioreactor.The new bioreactor can effectively improve various shortcomings of the the first generation.of bi-directional rotation and perfusion microgravity bioreactor and greatly improve the density, survival rate and function in vitro cultured human liver cells.It is expected to become a new generation of bioartificial liver bioreactor. |
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