【作者】 周焕城；

【导师】 高毅； 汪艳；

【作者基本信息】 南方医科大学 ， 肝胆外科， 2011， 博士

【摘要】 第一部分新型双向旋转灌注微重力生物反应器研制研究背景肝功能衰竭是各种肝病的终末期表现。患者病情危重,病死率高。肝脏移植手术是目前公认有效的治疗方法。但由于供体缺乏、高额的治疗费用以及患者需长时间服用免疫抑制剂等原因,极大地限制了肝移植手术的广泛开展。以体外培养肝细胞为基础的生物人工肝等治疗手段的出现,有望使部分患者肝功能自行恢复或顺利完成肝移植手术。然而,如何合理设计新型生物反应器,实现体外肝细胞的大规模培养及其培养过程中肝细胞功能与活率的有效维持,仍是目前强烈限制生物人工肝发展的瓶颈问题,也是目前亟待解决的重要课题。生物反应器是整个BAL的核心部分,其性能将直接关系到生物人工肝的支持效果。理想的生物人工肝生物反应器应满足如下条件：1.为肝细胞提供良好生长和代谢环境；2.实现有效的营养物质、氧气及代谢产物双向物质传输；3.保护肝细胞免受体内的免疫损伤；4.可进一步扩大培养规模,满足临床治疗需求；5.最大可能减小反应器内的培养死腔。为此,国内外学者研制出了各种结构形态的生物反应器,主要有四种模式：中空纤维型、单层平板培养型、灌流平板／支架型、包裹式微球／悬液或柱状反应器及其它结构复杂的复合式生物反应器等。虽然经过二十余年发展,已有部分生物人工肝生物反应器已进入临床实验,但目前仍未有一种理想的生物反应器可充分满足临床运用需要。大量研究表明,由美国航空航天局(NASA)设计的50ml小体积旋转式细胞培养系统(The Rotary Cell Culture System,以下简称RCCS-1),由于其独特的低剪切力、高效物质交换,持续模拟微重力培养环境可增强体外培养细胞与细胞间接触,形成三维结构样组织,促进细胞增殖分化,改善及维持体外培养细胞功能,目前已成功广泛地运用于胚胎干细胞、角膜细胞、骨骼肌细胞、成骨细胞等多个组织工程领域中。为进一步深入研究微重力生物反应器作为一种新型生物人工肝生物反应器的可行性本课题组引进了美国航空航天局(National Aeronautics and Space Administration简称NASA)设计的500ml体积的新型旋转灌注微重力生物反应器(Rotary Culture MWTM,简称RCMW),但经过反复的实验研究表明,美国航空航天局生产的RCMW微重力生物反应器在设计上存在严重的缺陷,完全不能满足生物人工肝生物反应器的需求,为此,本课题拟通过对RCMW微重力生物反应器设计上存在的问题进行优化改进,设计出一种具有自主知识产权的新型双向旋转灌注式微重力生物反应器,为新一代生物人工肝生物反应器的发展开辟一个新的方向与思路。研究目的通过对RCMW微重力生物反应器设计上存在的问题进行优化改进,设计出了一种新型双向旋转灌注式微重力生物反应器。研究方法在美国NASA公司RCMW微重力生物反应器的基础上,通过内芯及循环模式优化构建,形成由流向控制器、细胞培养罐、培养液池、蠕动泵动力系统、膜肺、供气系统组成的新型双向旋转灌注微重力生物反应器系统。人肝细胞(CL-1)在RCMW微重力生物反应器(RCMW组)、外置氧合器RCMW微重力生物反应器(RCMW高氧组)及双向旋转灌注微重力生物反应器(双向反应器组)中,持续微载体三维培养7天,并通过MTT染色、细胞计数、培养上清中ALT、AST、白蛋白、尿素浓度等指标测定,比较各组肝细胞活力、数量和功能差异。计量资料以均数±标准差(x±s)表示,采用SPSS 17.0统计软件分析。数据采用重复测量的方差分析,每组不同时间点之间的分析采用单组的重复测量方差分析,同一时间点的不同组别之间采用单向方差分析,当方差不齐时采用Welch方法。多重比较当方差齐性时采用LSD方法,当方差不齐时采用Tamhane’sT2方法。检验水准a=0.05.研究结果美国NASA公司商品化RCMW微重力生物反应器存在微载体/细胞堵塞,双向物质交换效率低下及出现培养死腔等缺点；而改进后双向旋转灌注微重力生物反应器中双向物质交换效率明显提高,微载体/细胞均匀分布于整个培养罐中,不存在培养死腔。倒置显微镜下形态学及MTT细胞活力染色结果可见,双向反应器组人肝细胞(CL-1)的数量与活力均明显优于RCMW组及RCMW高氧组。生长曲线结果显示,RCMW组、RCMW高氧组及双向反应器组人肝细胞密度均为先升高后逐渐下降,分别于第2天、第3天、第5天达肝细胞密度峰值8.8±0.57×105个/ml、12.92±0.98×105个/ml、32.58±1.44×105个ml,且双向反应器组人肝细胞密度于第2至7天均明显高于RCMW组及RCMW高氧组,差异均有统计学意义(P<0.01)。肝细胞功能结果显示,RCMW组、RCMW高氧组及双向反应器组白蛋白、尿素合成功能均为先升高后逐渐下降,分别于第3天、第3天及第5天达峰值,且双向反应器组上清白蛋白、尿素浓度于第1至7天均明显高于RCMW组及RCMW高氧组,差异均有统计学意义(P<0.01)。此外,双向反应器组上清ALT、AST浓度于第1至7天均明显低于RCMW组及RCMW高氧组,差异均有统计学意义(P<0.01)。研究结论本研究通过对RCMW微重力生物反应器设计上存在的问题进行优化改进,成功设计出了一种新型双向旋转灌注式微重力生物反应器,可有效解决商品化RCMW微重力生物反应器中存在的各种设计缺点,大大提高体外培养人肝细胞的密度、活率及功能,有望成为新一代生物人工肝生物反应器。第二部分双向旋转灌注微重力生物反应器人肝细胞、肝星形细胞共培养研究研究背景如何能尽最大可能地模拟肝细胞体内生存的微环境,使肝细胞能持续大量增殖分化,保持较高生物活性,且较好地保持肝细胞的各种生物功能是目前限制生物人工肝体外肝细胞规模化培养发展的瓶颈问题。为此,人们不断改进肝细胞体外培养方法。体内肝脏细胞由肝实质细胞和肝窦内皮细胞、肝星形细胞及库否细胞等肝非实质细胞组成。它们在错综复杂的三维细胞外基质中,通过细胞-细胞与细胞-细胞外基质间的相互通讯,精确地发挥着各自特异的生物学功能。这些重要的体内生理调节作用对于体内肝细胞功能与活性具有重要的作用。目前很多肝细胞体外培养研究中都在尝试尽可能地模拟体内这种生理结构,并经过长期的研究已经表明,通过与库否细胞、人脐静脉内皮细胞及成纤维细胞等肝内外非实质细胞共培养,可显著增强并维持体外培养肝细胞的各种肝特异性功能。肝星形细胞位于肝叶Disse间隙内,可合成大量ECM、摄取并贮存维生素A。正常肝组织内星形细胞的数量虽然不多(约占肝小叶的1%),但其细胞表面细长突起和皱折大大增加了其与肝实质细胞微绒毛的接触面,从而形成以星形细胞为中介的三位一体的结构功能单位,称为星形细胞单位。将肝实质细胞和星形细胞混合培养可望获得与肝小叶相似的功能,为生物人工肝提供具有良好生理功能的肝细胞来源。本课题在前期工作中研制成功具有自主知识产权双向旋转灌注微重力生物反应器,与目前现存的其它类型生物反应器培养系统相比,有如下优点：低剪切力,对细胞损伤小：高效的双向物质交换：持续模拟微重力环境有利于细胞增值分化,有利于细胞与细胞间接触,形成三维结构样组织易于细胞规模化培养等优点,现为最大可能地模拟肝细胞体内生存的微环境,使肝细胞能持续大量增殖分化,保持较高生物活性,较好地保持肝细胞功能,本课题拟在前期双向旋转灌注微重力生物反应器微载体人肝细胞的基础上,进一步进行微载体人肝细胞、人肝星形细胞三维共培养研究。研究目的通过人肝细胞、肝星形细胞微载体三维共培养进一步提高肝细胞数量、活力及功能。研究方法人肝细胞在微载体三维静置培养(微载体静置单培养组)、微载体人肝细胞、肝星状细胞三维静置共培养(微载体静置共培养组)、双向旋转灌注微重力生物反应器微载体三维培养(双向反应器单培养组)及双向旋转灌注微重力生物反应器微载体人肝细胞、肝星状细胞三维共培养(双向反应器共培养组)四种不同培养条件下连续培养7天。通过扫描电子显微镜、倒置显微镜观察及MTT染色比较各组肝细胞生长的形态、细胞活力差异,并通过细胞计数、培养上清中ALT、AST、白蛋白、尿素浓度等指标测定,比较各组肝细胞数量和功能差异。计量资料以均数±标准差(x±s)表示,采用SPSS 17.0统计软件分析。数据采用重复测量的方差分析,每组不同时间点之间的分析采用单组的重复测量方差分析,同一时间点的不同组别之间采用单向方差分析,当方差不齐时采用Welch方法。多重比较当方差齐性时采用LSD方法,当方差不齐时采用Tamhane’sT2方法。检验水准a=0.05.研究结果倒置显微镜、扫描电子显微镜及MTT细胞活力染色结果可见,微载体静置共培养组及双向反应器共培养组中,多个微载体间借“细胞桥”相互聚集在一起组成较大的微载体细胞组织块,细胞数量及活力均明显优于相应的微载体静置单培养组及双向反应器单培养组。生长曲线结果显示,微载体静置单培养组、微载体静置共培养组、双向反应器单培养组及双向反应器共培养组四组肝细胞密度均为先升高后逐渐下降,四组分别于第3天、第4天、第5天及第5天达峰值：8.22±0.60×105/ml、13.80±1.12×105/ml、32.62±2.51×105/ml和39.38±3.67×105/ml双向反应器共培养组肝细胞峰值密度明显高于其它三组,差异具有统计学意义(P<0.01)。微载体静置共培养组肝细胞密度于第3至7天均明显高于微载体静置单培养组,差异具有统计学意义(P<0.05)。双向反应器共培养组肝细胞密度于第2至7天均明显高于双向反应器单培养组,差异具有统计学意义(P<0.05)。双向反应器共培养组肝细胞密度于第1至7天均明显高于微载体静置共培养组,差异具有统计学意义(P<0.01)。肝细胞功能结果显示,双向反应器共培养组上清尿素、白蛋白峰值浓度明显高于其它三组,差异具有统计学意义(P<0.01)。微载体静置共培养组上清尿素浓度、白蛋白浓度于第2至7天均明显高于微载体静置单培养组,差异具有统计学意义(P<0.05)。双向反应器共培养组上清尿素浓度、白蛋白浓度于第2至7天均明显高于双向反应器单培养组,差异具有统计学意义(P<0.05)。双向反应器共培养组上清尿素浓度、白蛋白浓度于第1至7天均明显高于微载体静置共培养组,差异具有统计学意义(P<0.01)。研究结论1、人肝细胞、肝星形细胞共培养能进一步提高肝细胞的功能表达、延长细胞生长时间。2、双向旋转灌注微重力生物反应器微载体人肝细胞、肝星状细胞三维共培养是维持肝细胞大量增殖分化并保持肝细胞功能一种较为理想的体外肝细胞培养模式。第三部分肝细胞规模化培养保护液的研制研究背景肝细胞是生物人工肝(BAL)系统的核心。生物人工肝(BAL)对肝功能衰竭病人的肝支持作用完全依赖于所用肝细胞的特异性生物学功能。目前认为,生物人工肝要达到理想支持效果至少需要1010以上数量级的细胞,而在保障细胞活性的条件下,肝细胞数量越多,其支持及治疗效果将越佳,因此,肝细胞体外大规模培养技术已成为生物人工肝技术发展的核心技术。虽然肝细胞体外大规模培养技术已取得了许多重大的进展,如新型生物反应器研制及共培养等培养模式优化等,但肝细胞在体外大规模培养过程中仍容易受到氧气营养物质供应不足、代谢产物蓄积等各种因素的损伤,从而导致肝细胞的活性及功能下降。目前研究表明,在肝细胞体外大规模培养过程中,各种损伤因素所引起的肝细胞凋亡正是导致肝细胞活性与功能下降的主要原因。为此,本课题拟通过研制肝细胞规模化培养保护液,有效减轻规模化培养中各种培养环境因素对肝细胞造成的损伤,并抑制肝细胞凋亡的发生,从而达到进一步提高并维持肝细胞规模化培养的密度和功能状态的效果。研究目的研制一种肝细胞规模化培养保护液配方,可在同等条件下有效减轻规模化培养中各种培养环境因素对肝细胞造成的损伤,以进一步提高肝细胞规模化培养的密度和功能状态。研究方法根据国内外研究成果及前期预实验,自行研制出肝细胞保护液配方,成分主要包括黄芪、丹参、果糖、胰岛素及维生素C等。人肝细胞在RCMW微重力生物反应器微载体培养(RCMW组)、RCMW微重力生物反应器微载体培养+肝细胞保护液(RCMW保护液组)、双向旋转灌注微重力生物反应器微载体培养(双向反应器组)及双向旋转灌注微重力生物反应器微载体培养+肝细胞保护液(双向反应器保护液组)四种不同培养条件下连续培养7天,通过倒置显微镜观察及MTT染色比较各组肝细胞生长的形态、细胞活力差异,并通过细胞计数、培养上清中ALT、AST、白蛋白、尿素等指标测定,比较各组肝细胞数量和功能情况。以评价上述保护液配方在RCMW及双向旋转灌注微重力生物反应器两种不同的大体积(500m1)生物反应器中的肝细胞保护效果。计量资料以均数±标准差(x±s)表示,采用SPSS 17.0统计软件分析。数据采用重复测量的方差分析,每组不同时间点之间的分析采用单组的重复测量方差分析,同一时间点的不同组别之间采用单向方差分析,当方差不齐时采用Welch方法。多重比较当方差齐性时采用LSD方法,当方差不齐时采用Tamhane’sT2方法。检验水准a=0.05.研究结果倒置显微镜下形态学及MTT细胞活力染色结果可见,RCMW保护液组及双向反应器组肝细胞数量及活力均明显优于相应的RCMW组双向反应器组。生长曲线结果及肝细胞功显示,RCMW组、RCMW保护液组、双向反应器组及双向反应器保护液组四组肝细胞密度均为先升高后逐渐下降,四组分别于第2天、第4天、第5天及第5天达峰值：8.82±0.59×107ml、13.60±1.00×107ml、32.62±2.68x 105/ml和39.9±3.08×105/ml。RCMW保护液组细胞密度于第3至7天均明显高于RCMW组,差异具有统计学意义(P<0.05)。双向反应器保护液组细胞密度于第2至7天均明显高于双向反应器组,差异具有统计学意义(P<0.05)。RCMW保护液组上清尿素、白蛋白浓度于第1至7天均明显高于RCMW组,差异具有统计学意义(P<0.05)。双向反应器保护液组上清尿素、白蛋白浓度于第1至7天均明显高于双向反应器组,差异具有统计学意义(P<0.05)。而RCMW保护液组ALT、AST浓度于第2至7天均明显低于RCMW组,差异具有统计学意义(P<0.05)。双向反应器保护液组ALT、AST浓度于第4至7天均明显低于双向反应器组,差异具有统计学意义(P<0.05)。研究结论本课题组研制的肝细胞规模化培养保护液,可在同等条件下有效减轻规模化培养中各种培养环境因素对肝细胞造成的损伤,以进一步提高肝细胞规模化培养的密度和功能状态。更多还原

【Abstract】 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更多还原