| Part ITitle:Development of a novel bi-directional rotation and perfusion microgravity bioreactor system.Background:Liver failure is the performance of a variety of end-stage liver disease. Patients with liver failure are in critical condition, and is associated with a high risk of mortality. Currently, the only effective long term treatment for liver failure is orthotopic liver transplantation (OLT). But the orthotopic liver transplantation (OLT) was greatly restricted due to the the shortage of organ availability, long term immunosuppression, and the high cost of transplantation. The development of bioartificial liver (BAL) which is based on the cultured hepatocytes is the alternative ways to temporarily support the failing liver until a compatible donor can be found (bridging-to-transplantation) or, ideally, until the liver naturally regenerates and recovers its full functions bridging-to-regeneration).However, the development of bioaitificial liver is now greatly restricted by the design of an idea novel bioreactor,in which large-scale cultured hepatocytes can maintain their functions and viability well. Therefore, the topic of structural optimization and modifications for the novel bioreactor suitable for BAL systems is both important and imminent for the development of bioartificial liver (BAL).It is well known that the bioreactor is the key device in a BAL, its performance is thus directly associated with the efficacy of the BAL. The ideal bioreactor should serve several main functions:1. provision of a good environment for growth and metabolism of hepatocytes; 2.adequate bidirectional mass transport between media and hepatocytes; 3.protection of hepatocytes from impairment of the host immune system;4. potential for scale-up to therapeutic levels; and 5. minimization of the invalid space or dead volume within the device. In terms of structure, BAL bioreactors are mainly categorized as:hollow fiber; flat plate and monolayer; perfused beds or scaffolds; beds with encapsulated or suspended cells; and other complicated configurations with compound bioreactors. Although there has been more than 2 decades of exploration in this field, some bioreactors are currently under clinical investigation, further enhancements in the current systems for satisfying the demand of clinical applications are still necessary for the success of this application.Extensive researches show the small volume 50ml Rotatory Cell Culture System (RCCS), introduced by NASA, has been widely and successfully applied in many tissue engineer fields, such as embryonic stem (ES) cells,keratocytes, skeletal muscle and osteoblast, because of it enables the growth and suspension of anchorage-dependent cells under a low-shear-stress, high-mass-transfer, and persistent simulated microgravity condition allowing for the formation of 3D organized tissue-like structures and thereby enhancing cell proliferation, differentiation and improving the function of the cultured cells.For further study the feasibility of microgravity bioreactor as a new bio-artificial liver bioreactor of a new generation, we purchased the 500ml rotating and perfusion microgravity bioreactor (Rotary Culture MWTM RCMW) from the National Aeronautics and Space Administration (NASA) in America. However, our repeated preliminary experiments results showed that the RCMW bioreactor has serious flaws in the design. Therefore, in the present study, we have developed a novel bi-directional rotation of perfusion bioreactor system with independent intellectual property rights by structural optimization and modification of the serious flaws in RCMW.Our bioreactor will give a new direction for the development of the bioartificial liver bioreactor.Objectives:To developed a novel bi-directional rotation and perfusion bioreactor system by structural optimization and modification of the serious flaws in RCMW.Methods:We have developed a bi-directional rotation and perfusion bioreactor system by optimizing of the inner core and circular model of the RCMW bioreactor. The bi-directional rotation and perfusion bioreactor system was comprised by a directional controller, a cell culture vessel, a culture media reservoir, peristaltic pumps, a ertro corporeal membrane oxygenation and the gas supplying system. We compared herein the viability, culture density and functional activities of human hepatocyte (CL-1) in different bioreactor system during a 7-day culture periods.:which include RCMW bioreactor system (RCMW group); RCMW bioreactor system with high oxygen concentration (high oxygen RCMW group)and the bi-directional rotation and perfusion bioreactor system(bidirectional bioreactor group). The evaluating indicator of the viability, culture density and functional activities of human hepatocyte (CL-1) include MTT staining, cell counting and the concentration of ALT, AST, albumin, urea in the culture media.Statistical analysis was performed by using SPSS 17.0.Results Results are reported as means±standard error. Repeated measurement analysis of variance rank tests were used to compare the 3 bioreactor system over the 7-day culture period, and to compare differences per day within 1 cell type. Significance was reached if P< 0.05. Prism version 4.0 (GraphPad Prism, San Diego, CA) was used for graphical presentation of the data.Results:The RCMW bioreactor designed by NASA has serious flaws,includeing microcarrier accumulation,low exchange efficiency and dead culture space. The opmized bi-directional rotation and perfusion microgravity bioreactor system can achieve efficient two-way mass transfer, make cell/microcarrier evenly distributed throughout the culture bottles and reduce the dead cultivation space of the reactor. The quantity and viability of the human hepatocyte (CL-1) in bidirectional bioreactor group are much better than the RCMW group and high oxygen RCMW group according to the inverted microscope result and the MTT staining result. The growth curves of the human hepatocyte (CL-1) show that the cell density in RCMW group, high oxygen RCMW group and bidirectional bioreactor group,whose peak value reach 8.8±0.57×105/ml,12.92±0.98×105/ml,32.58±1.44×105/ml in day2,day3, day5 respectively, go up firstly, and then gradually drop from the top together. And the cell density in bidirectional bioreactor group is significantly higher than RCMW group and high oxygen RCMW group from day2 to day7 (P<0.01). The functional results show that the albumin and urea concentration in RCMW group, high oxygen RCMW group and bidirectional bioreactor group,whose peak value reach in day3,day3, day5 respectively, also go up firstly, and then gradually drop from the top together. And the albumin and urea concentration in bidirectional bioreactor group is significantly higher than RCMW group and high oxygen RCMW group from day2 to day7 (P<0.01).Besides, the concentration of ALT and AST in bidirectional bioreactor group is significantly lower than RCMW group and high oxygen RCMW group from day2 to day7(P<0.01)Conclusion:We have developed a novel bi-directional rotation and perfusion bioreactor system by structural optimization and modification of the serious flaws of RCMW., which offers new perspectives in bioartificial liver bioreactor.Part IITitle:Study for 3D coculture of human hepatocyte and satellite cell in novel bidirectional rotation and perfusion bioreactor systemBackground:People has been continuously improving the cell culture model for simulating the in vivo microenvironment of liver tissue and providing consistent and sufficient support for long-term maintenance of liver cell proliferation, viability and various liver function, which is greatly restricting the development of bioartificial liver.In a normal liver tissue, he hepatic parenchyma consists of parenchymal cells and non parenchymal cells such as sinusoids endothelial cells, stellate cells and Kupffer cells, and so on. They accurately present their biological functions under cell-cell, cell-extra-cellular matrix(ECM), and cell-microenvironment interactions in 3D complicated extra-cellular matrix(ECM).It has been widely explored and acknowledged for their major role in the maintenance of hepatic functions. Many studies tried to simulate the physiological organization of liver tissue by recruitment of the nonparenchymal cells NPCs into the liver function unit such as kupffer cells, human umbilical vein endothelial cells (HUVECs) and fibroblasts.It has been shown that the various hepatic functions can be remarkably enhanced in the coculture system. Hepatic stellate cells,which lie in the perisinusoidal space of Disse in hepatic lobule, can store vitamin A and synthesize a large amount of extracellular matrix (ECM).Although the quantity of the stellate cells in the normal hepatic tissue is small (about 1% in hepatic lobule), the stellate cells contact can widely widely with the microvilli of the hepatic parenchymal cells by vimineous corrugates on the surface, forming a structural functional unit (stellate cells unit). It is expected to supply good functional hepatocytes for the bioartificial liver by coculturing with the stellate cells.In the previous study,we have developed a novel bi-directional rotation and perfusion bioreactor system with independent intellectual property rights. It has the following advantages compared with the other existing bioreactor:low-shear-stress, high-mass-transfer, and persistent simulated microgravity condition allowing for the formation of 3D organized tissue-like structures and thereby enhancing cell proliferation, differentiation and improving the function of the cultured cells. In the present study, we are plan to further enhance proliferation, viability and liver-specific functions of hepatocytes by 3D co-culturing with stellate cells in our novel bi-directional rotation and perfusion bioreactor system.Objection:To further enhance proliferation, viability and liver-specific functions of hepatocytes by 3D co-culturing with stellate cells.Mehtods:We compared herein the viability, culture density and functional activities of human hepatocyte (CL-1) in different culture system during a 7-day culture period.: which include static 3D culture (static 3D culture group); static 3D coculture with stellate cells (static 3D coculture group)and culture in the bi-directional rotation perfusion bioreactor system(bidirectional bioreactor group) and 3D coculture with stellate cells in the bi-directional rotation perfusion bioreactor system(bidirectional bioreactor coculture group).The evaluating indicator of the viability, culture density and functional activities of human hepatocyte (CL-1) include MTT staining, SEM,cell counting and the concentration of ALT, AST, albumin, urea in the culture media.Results:According to the inverted microscope result and the MTT staining result:a number of microcarriers aggregate into big microcarrier/cell conglomerations by "cell bridge" in static 3D coculture group and bidirectional bioreactor coculture group, in which the quantity and viability of the human hepatocyte (CL-1) are much better than the corresponding static 3D culture group and bidirectional bioreactor group. The growth curves of the human hepatocyte (CL-1) show that the cell density in static 3D culture group, static 3D coculture group, bidirectional bioreactor group and bidirectional bioreactor coculture group, whose peak value reach 8.22±0.60×105/ml,13.80±1.12×105/ml,32.62±2.51×105/ml and39.38±3.67×105/ml in day3, day4, day5 and day5 respectively, go up firstly, and then gradually drop from the top together. The peak cell density value in bidirectional bioreactor coculture group is significantly higher than other 3 group (P<0.01). And the cell density in static 3D coculture group is significantly higher than the static 3D culture group from day2 to day7 (P<0.05) the cell density in bidirectional bioreactor coculture group is significantly higher than the bidirectional bioreactor group from day2 to day7 (P<0.05)The peak value of albumin and urea concentration in bidirectional bioreactor coculture group is significantly higher than other 3 group (P<0.05). And albumin and urea concentration in static 3D coculture group is significantly higher than the static 3D culture group from day2 to day7 (P<0.05) the c albumin and urea concentration in bidirectional bioreactor coculture group is significantly higher than the bidirectional bioreactor group from day2 to day7 (P<0.01)Conclusion: l.The proliferation, viability and liver-specific functions of the hepatocytes can be further enhanced by 3D co-culturing with stellate cells.2.Hepatocyte 3D coculture with stellate cells in our bi-directional rotation perfusion bioreactor system is a effective culture model for maintaining proliferation, viability and liver-specific functions of the cultured hepatocytes. According to the domestic and international research and preliminary experiment, we have developed a hepatocyte protective reagent, which comprise astragalus, salvia miltiorrhiza, fructose, insulin and vitamin C and so on. We compared herein the viability, culture density and functional activities of human hepatocyte (CL-1) in different bioreactor and culture condition during a 7-day culture period.:which include RCMW bioreactor system (RCMW group); RCMW bioreactor system administered with hepatocyte protective reagent (RCMW protective reagent group)and bi-directional rotation perfusion bioreactor system (bidirectional bioreactor group) and bi-directional rotation perfusion bioreactor system administered with hepatocyte protective reagent (bidirectional bioreactor protective reagent group).The evaluating indicator of the viability, culture density and functional activities of human hepatocyte (CL-1) include MTT staining, SEM,cell counting and the concentration of ALT, AST, albumin, urea in the culture media.Results:According to the inverted microscope result and the MTT staining result:the quantity and viability of the human hepatocyte (CL-1) in RCMW Protective Reagent group and Bidirectional Bioreactor Protective Reagent group are much better than the corresponding RCMW group and Bidirectional Boreactor group. The growth curves of the human hepatocyte (CL-1) show that the cell density in RCMW group, RCMW Protective Reagent groupp, Bidirectional Bioreactor group and Bidirectional Bioreactor Protective Reagent group, whose peak value reach 8.82±0.59×105/ml,13.60±1.00×105/ml,32.62±2.68×105/ml and 39.9±3.08×105/ml in day2, day4, day5 and day5 respectively, go up firstly, and then gradually drop from the top together. And the cell density, albumin and urea concentration in RCMW Protective Reagent group is significantly higher than the RCMW group from day2 to day7 (P<0.01).The cell density, albumin and urea concentration in Bidirectional Bioreactor Protective Reagent group is significantly higher than the Bidirectional Bioreactor group from day2 to day7 (P<0.01). Contrarily, the ALT and AST concentration in RCMW Protective Reagent group is significantly lower than the RCMW group from day2 to day7 (P<0.01).The ALT and AST concentration in Bidirectional Bioreactor Protective Reagent group is significantly lower than the Bidirectional Bioreactor group from day2 to day7 (P<0.01)Conclusion:We have developed a novel protective reagent, which is able to effectively reduce the hepatocyte damage from various environmental factors, inhibit the cell apoptosis, and at last improve the cell density and function. in the same culture condition... |
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