生物人工肝人肝细胞系进展

大多数生物人工肝 (bioartificialliver,BAL)装置以原代猪肝细胞作为生物成分 ,然而 ,由于异种移植相关危险性 ,猪肝细胞终究不是最佳选择。人肝细胞系无论哪方面都具有比动物肝细胞要好的特性 ,所以是应用于BAL的候选细胞。肿瘤来源细胞系C3A和HepG2具有多种肝细胞功能 ,但缺乏一些特异的肝功能 ,如氨解毒和尿素生成等。永生化肝细胞可能提供更好的BAL应用前景。另一有希望的方法是应用胚胎或成人干细胞。啮齿动物干细胞已经可在体外定向分化为肝细胞 ,这些体外诱导方法可能也适用于人干细胞。但是 ,在人肝细胞系和分化的干细胞成功…     

生物人工肝人肝细胞系进展

大多数生物人工肝(bioartificial liver，BAL)装置以原代猪肝细胞作为生物成分，然而，由于异种移植相关危险性，猪肝细胞终究不是最佳选择。人肝细胞系无论哪方面都具有比动物肝细胞要好的特性，所以是应用于BAL的候选细胞。肿瘤来源细胞系C3A和HepG2具有多种肝细胞功能，     

用流式细胞仪测定人肝细胞系DNA指数值作为其生物功能筛选指标的研究

目的　了解DNA指数 (DI)与人肝细胞系生物功能之间的关系。探讨DI值可否作为人工肝生物材料的初步筛选指标。方法　收集国内 9株人工肝细胞系 ,分别编号为细胞系 1- 9(CL1-9)。分别培养后获取细胞。取正常人全血分离淋巴细胞 ,作为正常二倍体对照。以流式细胞仪(FCM)测定 9株人肝细胞系DI值作为分化指标 ,判断人肝细胞系的分化情况。人肝细胞系分别接种1.2× 10 6个细胞 ,收集其培养生长旺盛、生物功能良好的第 5天时的培养上清液 ,测定上清液中的尿素、人白蛋白和安定含量 ,计算细胞系尿素合成、人白蛋白合成和安定转化量。进行DI值和肝功能指标间的相关性分析。CX7自动生化分析仪测定尿素浓度 ;用人白蛋白放免试剂盒经改良后采用放射免疫抗原竞争法测定人白蛋白浓度 (人白蛋白与牛血清白蛋白无交叉反应 ) ;培养上清中另加入 4 μg/ml安定作肝细胞药物代谢功能指标 ,提取后以 75 2型紫外分光光度计测定安定含量。采用双变量相关与回归分析方法进行统计学分析。结果　人肝细胞系DI值与人肝细胞系尿素合成量、人白蛋白合成量及安定转化量均呈线性相关 ,相关系数分别为 - 0 .94 37、- 0 .8795、- 0 .9386。结论　CL1分化程度最高 ,生物功能最好 ;DI值可以作为人工肝生物材料的初步筛选指标     

人肝细胞系L-02与聚丙烯生物杂化界面构建的初步研究

目的　建立与人肝细胞相容的聚丙烯生物杂化界面 ,为用聚丙烯中空纤维管构建生物人工肝反应器奠定基础。方法　通过化学接枝的方法在聚丙烯表面引入聚丙烯酰胺形成聚合反应 ,并对人肝细胞系L 0 2在其表面的生长特点进行检测评价。结果　聚丙烯膜的静态水相接触角由接枝前的 (72± 5 )°降低为接枝改性后的 (30± 4 )°,明显提高了接枝改性聚丙烯膜的亲水性 ,并通过简单的静止培养使人肝细胞系L 0 2在其表面呈球形聚集体生长 ,提高了培养密度和活性。结论　在聚丙烯表面接枝聚丙烯酰胺可初步建立良好的人肝细胞系L 0 2与聚丙烯生物杂化界面 ,此肝细胞球形聚集体培养方法较为简单     

Cytodex-3微载体高密度培养人肝细胞系CL-5

目的采用微载体进行高分化的人肝细胞系CL－5的高密度培养以作为生物人工肝的生物材料。方法进行CL－5的微载体均匀搅拌培养,于培养的1、3、5、7、9天进行细胞计数及培养上清人白蛋白浓度检测,并在显微镜下动态观察细胞生长情况。结果细胞密度于培养的第七天达最高峰为1.98×10~9／L,同时人白蛋白的分泌亦最高,为54.79μg。结论实验表明使用Cytodex-3微载体培养CL-5肝细胞系,可达到较高的密度,并且具有一定的分泌功能。     

微载体培养人肝细胞系作为人工肝生物材料的研究

目的 为提高人肝细胞系的培养效率和细胞获取数量。方法 本研究采用微载体细胞培养技术进行了人肝细胞系CL-1的高密度培养，动态观察细胞生长，检测肝细胞特异性功能变化。结果 CL-1在微载体Cytodex-3上生长良好，于培养的第7天达到高峰，细胞数量为2.13×10^8／100ml，白蛋白合成量71．23μg／100ml．尿素合成量23．32mg／100ml，安定转化量619．7μg／100ml，与CL-1普通培养结果比较，细胞产量是普通培养的49．3倍．而白蛋白合成量、尿素合成量及安定转化量则分别是普通培养的39．8倍、41．6倍和33．3倍。结论 微载体培养CL-1可提高培养效率和细胞数量．且具有较好的功能，微载体高密度培养CL-1可作为组合型人工肝的生物材料。     

微载体培养人肝细胞在混合型生物人工肝系统上的应用

目的　探讨微载体培养L 0 2人肝细胞株应用于混合型生物人工肝系统 (HBLS)及其治疗实验效果。方法　采用微载体Cytodex 3培养人肝细胞 ,经简易体外灌流实验后装配成HBLS ,用于治疗急性肝衰犬 ;观察模型犬的临床体征、生存时间、生化指标以及死亡后各脏器的病理检查。结果　HBLS治疗组和对照组的平均生存时间分别为 (7 9± 2 3)h和 (3 4± 1 4 )h ,差异具有显著性 (P <0 0 5 ) ;空舱对照组生化指标呈现进行性恶化趋势 ,而HBLS组治疗过程中 ,FBI上升 ,TBIL、ALT、AST、LDH、PT指标呈下降趋势 ;肝组织的病理显示治疗组较对照组肝细胞变性坏死程度减轻。结论　微载体培养L 0 2人肝细胞株成功应用于HBLS ,能改善急性肝衰犬的状态 ,延长生命 ,具有一定的安全性和有效性。     

微载体培养L02人肝细胞应用于生物人工肝的初步研究

Ｌ0 2人肝细胞株组织学来源为人正常肝组织 ,具备一定正常肝细胞的生物功能。我们将高密度微载体培养的Ｌ0 2人肝细胞聚集体加入中空纤维生物反应器 ,构建简易体外生物人工肝装置并进行初步的应用研究。一、材料与方法1.材料 :Ｌ0 2人肝细胞株购于中国科学院上海细胞生物学研究所 ;Ｃｙｔｏｄｅｘ3微载体为Ｐｈａｒｍａｓｉａ公司产品 ;聚羟乙基异丁烯酸 (Ｐｏｌｙ ＨＥＭＡ )及ＤＭＥＭ、ＨａｍｓＦ12培养基均为Ｓｉｇｍａ公司产品。2 .Ｌ0 2人肝细胞的微载体培养 :( 1)取 1.4ｇＣｙｔｏｄｅｘ 3,水化处理[1] ;( 2 ) 12 %Ｐｏｌｙ ＨＥＭＡ…     

人肝细胞的分离、培养和冻存

人肝细胞是生物人工肝的最理想细胞来源，决定其治疗效果。本文就人肝细胞分离、培养及冻存进行综述。     

Microencapsulated Hepatocytes for Bioartificial Liver Support

Free hepatocytes, harvested from normal rat livers by portal vein collagenase perfusion, were encapsulated within alginate-polylysine membranes and served as a liver support system. The encapsulated hepatocytes remained viable and were able to synthesize protein for up to 3 weeks in culture. Allografts of encapsulated hepatocytes replaced the function of a damaged liver and reduced the mortality rate among rats with galactosamine-induced fulminant hepatic failure.     

Development of a Bioartificial Liver Using Isolated Hepatocytes

Abstract: Severe liver disease is very often life-threatening and dramatically diminishes quality of life. Liver support systems based on detoxification alone have been proved ineffective because they cannot correct biochemical disorders. An effective artificial liver support system should be capable of carrying out the liver's essential processes, such as synthetic and metabolic functions, detoxification, and excretion. It should be capable of sustaining patients with fulminant hepatic failure, preparing patients for liver transplantation when a donor liver is not readily available (i.e., bridge to transplantation), and improving the survival and quality of life for patients for whom transplantation is not a therapeutic option. Recent advances in cell biology, tissue culture techniques, and biotechnology have led the way for the potential use of isolated hepatocytes in treating an array of liver disorders. Isolated hepatocytes may be transplanted to replace liver-specific deficiencies or as an important element of an auxiliary hybrid, bioartificial extracorporeal liver support device, which are important therapeutic applications for treating severe liver disease. Recently, several hepatocyte-based liver support systems have been proposed. Although there is no current consensus on its eventual design configuration, the hollow fiber hepatocyte bioreactor shows the greatest promise. Furthermore, application of tissue engineering technology, based on cellsurface interaction studies proposed by our group and others, has enhanced interest in the development of highly efficient hybrid, bioartificial, liver support devices.     

First Report of Cryopreserved Human Hepatocytes Based Bioartificial Liver Successfully Used as a Bridge to Liver Transplantation

Cryopreserved human hepatocytes could be the best type of cells to be used in a bioartificial liver (BAL) device due to reduced biosafety and biocompatibility risks. Banking of primary human hepatocytes, obtained from livers unwanted for transplantation at harvesting, could be used as a source of human liver cells for BAL treatment. We describe herein for the first time the case of a patient affected by fulminant hepatic failure (FHF) due to acute HBV infection that was successfully bridged to emergency liver transplantation by BAL treatment using cryopreserved primary human hepatocytes. The use of cryopreserved primary human hepatocytes as the biological part of the BAL device has never been described before and might be considered as a possible alternative to xenogenic material or human tumoral cell lines due to reduced biosafety and biocompatibility risks.     

Development of a Bioartificial Liver with Sandwiched-Cultured Hepatocytes Between Two Collagen Gel Layers

Abstract: We have been developing a multiplate type of bioartificial liver (BAL) using primary cultured hepatocyte monolayers since 1987. This BAL has been shown to prolong the survival time of anhepatic dogs and rabbits. Initially, hepatocytes were cultivated on collagen-coated plates. To increase the multiplated BAL function, a sandwich cultivation method was employed in which rat hepatocytes were cultured between two collagen gel layers and were then evaluated in both stationary and perfusion cultures. In the stationary culture, the sandwich method showed a higher activity in urea synthesis than in the other culture methods (culture on a collagen coating, culture on a collagen gel, and culture between a collagen coating and a collagen gel) for 14 days. In the perfusion culture, a BAL housing cultured hepatocytes (6.5 times 10 cells) in the sandwich system showed urea synthesis activity ranging from 17.5 to 22.6 μg/2 times 10⁶ cells/90 min. This activity was maintained for 5 days in the perfusion culture. The sandwich type of cultivation is applicable to the multiplated BAL.     

Packed-Bed Type Reactor to Attain High Density Culture of Hepatocytes for Use as a Bioartificial Liver

Abstract: In an attempt to develop a bioartificial liver using cultured hepatocytes, we investigated the short-term and long-term viability and metabolic functions of hepatocytes cultured in a new type of packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Perfusion culture experiments using this reactor, as well as monolayer cultures using conventional collagen-coated Petri dishes as control experiments, were performed. It was found that the highest density of immobilized hepatocytes attained with PVF resin was on the order of 10⁷ cells/cm³ PVF and that hepatocytes cultured in this type of module for up to a week showed a sufficient level of liver-specific metabolic functions, such as ammonium metabolism, urea-N synthesis, and albumin secretion, to be comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising means to develop a bioartificial organ using hepatocytes.     

Bioartificial Liver Using Hepatocytes on Biosilon Microcamers: Treatment of Chemically Induced Acute Hepatic Failure in Rats

An artificial liver support procedure based on hemoperfusion via hepatocytes cultured on microcarriers is described. The efficiency of the system was assessed by the survival rate of rats treated with either lethal dosage of 7% CCl4 [30 ml/kg body weight (b.w.)] or D-galactosamine (2.5 g/kg b.w.). In CCl4-treated rats, hemoperfusion via empty microcarriers (n = 16) revealed no surviving animals, whereas the use of the bioartificial liver (n = 11) resulted in 80% (p less than 0.01) and 60% (p less than 0.05) survival 48 and 168 h after hepatotoxin, respectively. For the same time periods, the survival rate in D-galactosamine-intoxicated rats after hemoperfusion with hepatocytes (n = 20) was approximately 60% (p less than 0.05) and was only 5% in those of rats treated with empty microcarriers (n = 20). Sublethal dosage of 7% CCl4 (15 ml/kg b.w.) caused 25% mortality and prolonged (48 h) increase of activity of the liver enzymes and bilirubin levels in the serum of surviving animals. In these rats (n = 8) at the end of 3 h of hemoperfusion via hepatocytes, the bilirubin concentration decreased by 45% as compared with the control group (n = 6) treated with empty microcarriers. Moreover, by 48 h after intoxication, the use of the bioartificial liver resulted in more than a three-fold decrease in glutamate-oxaloacetate transaminase and a 10-fold decrease in glutamate-pyruvate transaminase serum activity as well as a fivefold decline in total and a ninefold decline in conjugated bilirubin levels as compared with the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Auxiliary Liver Transplantation and Bioartificial BridgingProcedures in Treatment of Acute Liver Failure

The aim of this review is to discuss the place of auxiliary liver transplantation (ALT) and other cellular based bridging procedures such as hepatocyte transplantation, ex vivo liver perfusion, and bioartificial livers, in the treatment of acute liver failure, vis à vis conventional orthotopic liver transplantation. Hepacocyte transplantation, and ex vivo pig or human liver perfusion are still experimental procedures. Bioartificial livers using human tumoral hepatocytes or porcine hepatocytes have been used in clinical situations as a bridge to transplantation, i.e. to gain the time required to find a high-quality graft for conventional or auxiliary liver transplantation. None of these techniques have yet proved capable of keeping a patient alive long enough for the native liver to recover. Conversely, ALT has been shown to be effective in the treatment of acute liver failure and now appears to be a satisfactory bridging procedure pending native liver (NL) regeneration. We report personal experience of 18 ALT procedures performed in 17 patients between October 1992 and December 1999. The ALT procedure was indicated when patients met criteria for conventional transplantation; it was ultimately selected when a fresh frozen biopsy of the NL did no show any fibrosis. Six patients died within the first 2 postoperative months. The remaining 11 patients are alive, with a follow-up ranging from 2 to 7 years. Regeneration of the NL occurred in 11 of the 17 patients (65%) and in 8 of the 11 survivors (72%), 6 of whom have permanently stopped immunosuppressive therapy. We conclude that liver failure should no longer be handled outside centers where all types of transplantation can be offered, and where innovative therapies such as hepatocyte transplantation and extracorporeal liver-assist devices are being developed and evaluated.     

Isolation and Culture of Human Hepatocytes from Resected Liver Tissue as a Bioreactor for a Hybrid Artificial Liver

Abstract: To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated. Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined. The yield of isolated hepatocytes obtained by Method 2 (13.31 times 10⁶ cellslg of liver) was significantly higher than that by Method 1 (0.94 times 10⁶). The warm ischemia time (0–90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes. The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days. These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.     

Bioartificial Liver Support System Using Porcine Hepatocytes Entrapped in a Three-Dimensional Hollow Fiber Module with Collagen Gel: An Evaluation in the Swine Acute Liver Failure Model

The perfusion culture system of hepatocytes entrapped in a three-dimensional hollow fiber module with collagen gel is expected to realize an effective bioartificial liver (BAL) support system. The BAL module contained 5.4 billion porcine hepatocytes, which is approximately 7% of the total liver, supplied adequate metabolic functions, and improved the survival of the experimental acute liver failure swine. Improvement of the prothrombin time by the BAL treatment was not significant; however, the bleeding tendency was suppressed, which resulted in a significantly prolonged survival time of the animals in the BAL treatment group. A drastic up-regulation of the expression of the mRNA for plasminogen activator inhibitor 1 (PAI-1), which is known as an inhibitor for fibrinolysis, may explain the suppression of the bleeding tendency. These results support the efficacy of our BAL system for the treatment of acute liver failure.     

Generation of Humanized Liver Mouse Model by Transplant of Patient-Derived Fresh Human Hepatocytes

Some research groups have produced immunodeficient mice with human liver tissue as a model system for the analysis of drug metabolism and liver regeneration. Mouse models are important for research and development of drugs and vaccines for viral infections. Recent progress in developing humanized mouse models permits studies of adaptive immune responses, innate host responses, and therapeutic approaches for several liver diseases of viral etiology. In this study, we generated a humanized liver mouse model by transplant with fresh patient-derived hepatocytes (1 × 10⁶ cells/mouse, intrasplenic injection) into preconditioned (50 mg/kg ganciclovir, intraperitoneal injection) mice (herpes simplex virus type 1 thymidine kinase [TK] transgene expressed within the liver of a highly immunodeficient mouse strain [NOG]). Successful reconstitution of human hepatocytes in TK-NOG mouse liver tissues was observed with a strong proliferation of human cells in a time-dependent manner, using cytokeratin 8/18 stain. Similarly, we detected significantly increased human albumin levels in TK-NOG mouse liver tissue and blood sera on immune staining and enzyme-linked immunosorbent assay. Therefore, this humanized liver mouse model provides a biomedical tool for studying human liver physiology, drug metabolism, and liver pathogenesis of viral etiology or liver regeneration.