生物人工肝支持系统生物反应器的建立及体外转流试验

目的 探讨由微载体培养的L－02人肝细胞和中空纤维舱构成的生物反应器的生物效能。方法 采用微载体培养高浓度L－02人肝细胞，同时使用中空纤维型生物反应器和血泵等共同构成生物人工肝系统。在体外转流试验中观察循环液中游离胆红素、葡萄糖、白蛋白、谷草转氨酶浓度的变化以及实验对肝细胞的影响等。结果 L－02肝细胞能很好地粘附于cytodex 3微载体上形成微载体诱导的肝细胞聚集体；移入生物反应器内进行体外循环，4h后可见循环液中游离胆红素和葡萄糖浓度显著降低，分别为27.1μmmol/L和1.75mmol/L；白蛋白含量明显增加，为15．21mg/L；肝细胞仍有较高活力达75％。结论 本实验所建立的生物反应器作为生物人工肝系统的核心组件具备一定的生物合成和解毒代谢功能，为进一步建立混合型生物人工肝支持系统奠定了基础。     

猪内源性逆转录病毒透过生物人工肝的影响因素

目的 病毒安全性是猪源性生物人工肝(BAL)不可忽略的安全问题.本研究旨在探讨影响BAL中猪内源性逆转录病毒(PERV)透过的因素.方法 构建双循环的新型猪源性BAL.两循环间采用膜孔径为10 nm、20 nm、30 nm和35 nm的血浆成分分离柱以及500 nm膜孔径的血浆过滤柱分隔两循环.反应器内填充共培养的猪肝细胞-骨髓间充质肝细胞或原代猪肝细胞并连续循环72 h.每12 h抽取内、外循环内培养液检测PERV RNA、逆转录酶(RT)活性以及其体外感染性.结果 血浆过滤柱组中,内外循环内PERV检测结果完全相同,提示500 nm血浆滤过柱对PERV并无阻隔作用.在血浆成分交换柱各组中,各时间点内循环中均能检出RNA和RT活性,但是外循环中并未检出RT活性,而且随着膜孔径减小,RNA的检出时间逐渐延迟,且内循环RNA水平逐渐增加.各培养液均未发现能够体外感染HEK293细胞.结论 膜孔径、循环时间以及内循环病毒浓度是影响BAL中PERV透过的主要原因.     

新型编织型生物反应器内大量乳猪肝细胞的组织化培养

目的使反应器内培养的肝细胞能在较长时间保持肝细胞的特殊功能和活力,提高生物反应器的功效并为反应器内储存和运输肝细胞提供可能.方法将新生实验型小猪肝细胞与微载体共同培养,待肝细胞与微载体充分粘附形成微载体-球形聚集体后,将其置入新型编织型生物反应器的外腔,用培养液循环式人工毛细管培养系统进行培养,在培养液内加入氯化氨、醋胺酚检测生物反应器的转化功能,行无血清培养检测肝细胞的白蛋白的合成功能,观察肝细胞的酶漏出量.锥虫蓝染色观察肝细胞的活力,电镜观察其超微结构.结果生物反应器内培养的肝细胞在1周内能保持较高的活力,并保持着较高的氯化氨、醋胺酚的生物转化及白蛋白合成功能,酶的漏出量也少.肝细胞超微结构示内质网、线粒体等细胞器丰富,核内染色体分布均匀,肝细胞间的微绒毛形成胆小管样结构.结论肝细胞与微载体及肝细胞之间的聚集,形成直径大小不等的肝细胞-微载体球形聚集体,这种由肝细胞重新结合而成的聚集体类似于体内的肝组织结构,在新型编织型生物反应器内的中空纤维网架的支持下,肝细胞聚集体均匀地分散其中,为肝细胞的生长提供了一个类似体内内环境的三维空间,因而在无氧合器供氧的情况下,肝细胞也能在1周内保持较好的形态和功能.     

胶原凝胶包埋肝细胞体外培养和腹腔移植的研究

目的　研究改进肝细胞腹腔移植的方法。方法　用胶原凝胶包埋大鼠肝细胞 ,并在体外培养 ,测定培养液中总蛋白 (TP)和尿素氮 (BUN)水平。将胶原培养液与鼠肝细胞混合后注入大鼠腹腔 ,混合物在腹腔内形成凝胶并包埋肝细胞。用含酶的培养液将受者鼠腹腔内的移植肝细胞洗出 ,再置于无葡萄糖培养液中培养 ,测定培养液中葡萄糖含量。对照组处理过程与实验组相同 ,但不用胶原。结果　胶原凝胶包埋肝细胞组的TP、BUN和葡萄糖含量均明显高于对照组 (P <0 .0 1)。胶原培养液与肝细胞混合物在注入腹腔 2 4h后变为凝胶。结论　胶原凝胶可促进体外培养或腹腔移植的肝细胞生长 ;胶原培养液腹腔注射和腹腔内胶原凝胶包埋细胞法 ,使肝细胞的腹腔移植更简便、安全、实用。     

丹参对大鼠供肝冷保存缺血再灌注损伤中肝细胞凋亡的影响

目的 探讨丹参对大鼠原位肝移植供肝冷保存缺血再灌注损伤中肝细胞凋亡的影响.方法 将40只SD大鼠分为对照组、实验组及假手术组,建立原位肝移植模型.供肝灌注冷保存以4℃乳酸林格氏液为基液,实验组加丹参注射液(60 ml/L),对照组不加丹参.保存5 h后植入受体.移植后6 h处死大鼠取样,检测血浆中ALT、AST水平;采用TUNEL法检测移植术后肝细胞凋亡情况;流式细胞仪检测凋亡相关基因Bcl-2、FasL蛋白的表达;光镜下观察移植肝脏病理形态学的改变.结果 再灌注后实验组ALT、AST显著低于对照组.与对照组比较,实验组肝细胞凋亡指数明显下降(F=133.802,P<0.05);肝组织中Bcl-2蛋白表达增加(F=91.063,P<0.01);FasL蛋白表达无明显变化(F=1.329,P>0.05);实验组与对照组比较肝组织形态学的再灌注损害程度明显减轻.结论 丹参可通过促进抑制凋亡基因Bcl-2蛋白的表达来抑制冷保存再灌注导致的肝细胞凋亡,对原位肝移植肝脏的缺血再灌注损伤有保护作用.     

前列地尔预处理对移植肝TNF-α和细胞凋亡的影响

目的探讨前列地尔预处理对移植肝组织中肿瘤坏死因子α(TNF-α)水平的变化和细胞凋亡的影响。方法将30例终末期肝病患者随机分为实验组和对照组,对照组供体摘取后常规以4℃UW液灌注保存,实验组供体摘取后以UW液灌注同时以10μg前列地尔经门静脉灌注,4℃保存。实验组和对照组均在供肝恢复血流后3小时抽取外周静脉血检测肝功能,并取部分肝组织检测TNF-α水平和肝细胞凋亡指数。结果实验组肝组织中TNF-α水平低于对照组(P<0.05),实验组肝细胞凋亡指数低于和对照组。结论前列地尔预处理可减少供肝TNF-α释放,抑制炎症反应,减少细胞凋亡,减轻肝细胞损伤,对供肝有保护作用。     

肝细胞生长因子在肝星状细胞调控卵圆细胞凋亡中的作用

目的 探讨肝细胞生长因子(hepatocyte growth factor,HGF)在肝星状细胞(hepatic stellate cells,HSCs)调控卵圆细胞凋亡中的作用.方法 将健康雄性SD大鼠随机分为实验组、条件对照组和假手术组.三组大鼠均接受2-AAF灌胃,实验组和条件对照组进行2/3肝切除,而假手术组仅进行开关腹.于术后第0、1、3、6、9、12和15天提取HSCs,收集该细胞的条件培养液,将HGF受体抑制剂PHA665752(5 μmol/L)加入条件对照组的条件培养液中,用各时间点的条件培养液作用于LE6卵圆细胞系24 h,经PI/Annexin-Ⅴ染色后采用流式细胞术检测LE6细胞的凋亡情况.提取第9天条件培养液处理后的LE6细胞蛋白,采用Western Blotting检测Bim和cleaved-Casepase 3蛋白的表达.结果 经第6、9、12天条件培养液处理后,实验组LE6细胞的凋亡率显著低于假手术组和条件对照组(P＜0.05),其中第9天差异最显著,而且第9天实验组的Bim和cleaved-Casepase 3蛋白的表达量均低于假手术组和条件对照组(P＜0.05).结论 HGF可能通过降低Bim蛋白表达和抑制Caspase 3活性参与HSCs对卵圆细胞凋亡的负调控.     

生物人工肝的研究进展

肝移植是治疗急慢性肝功能衰竭最有效的方法,但是,由于供肝缺乏,必需寻求终未性肝病替代疗法.其中主要方法之一是体外生物人工肝支持系统,简称生物人工肝(bioartificial liver, BAL),发展至今已有40多年.临床试验证明BAL能促进肝功能衰竭病人的恢复,或过渡到肝移植.生物反应器是BAL的核心部件,生物反应器设计的主要目的在于保持肝细胞活力和功能,且不妨碍肝细胞营养及代谢产物的交换,同时还能起到治疗作用.细胞材料是BAL治疗基础,维持细胞活率和功能对BAL功效有决定性作用.本文就生物人工肝装置在实验和临床试验中的研究进展作一综述.……     

利多卡因对缺氧/复氧后肝细胞Bcl-2、Caspase-3蛋白表达及细胞凋亡的影响

目的探讨利多卡因预处理对肝细胞缺氧/复氧后Bcl-2、Caspase-3蛋白表达及细胞凋亡的影响。方法将体外培养的肝细胞分为三组:缺氧/复氧组(Ⅰ组)、利多卡因预处理组(Ⅱ组)和正常对照组(Ⅲ组),每组10份。检测肝细胞缺氧/复氧培养后细胞培养液中谷丙转氨酶(ALT)、谷草转氨酶(AST)浓度,肝细胞Bcl-2、Caspase-3蛋白表达、肝细胞凋亡率及超微结构变化。结果Ⅰ、Ⅱ组细胞培养液中ALT、AST浓度、肝细胞Caspase-3蛋白表达和肝细胞凋亡率较Ⅲ组均升高(P<0.05),透射电镜示肝细胞损伤,可见凋亡细胞;Ⅱ组细胞复氧后培养液中ALT、AST浓度、肝细胞Caspase-3蛋白表达和细胞凋亡率均低于Ⅰ组(P<0.05),而肝细胞Bcl-2蛋白表达较Ⅰ组升高(P<0.05),透射电镜观察也显示Ⅱ组肝细胞比Ⅰ组损伤轻微。结论利多卡因预处理可以在一定程度上减轻缺氧/复氧后肝细胞损伤,降低细胞凋亡率,保护机制可能与Bcl-2、Caspase-3蛋白表达有关。     

bFGF对大鼠缺血皮瓣琥珀酸脱氢酶含量和氧耗量的影响

为了观察碱性成纤维细胞生长因子（ｂＦＧＦ）对大鼠缺血皮瓣坏死率、琥珀酸脱氢酶（ＳＤＨ）含量和氧耗量的影响，选用Ｗｉｓｔａｒ大鼠１８只，随机分为对照组和实验组。制成背部皮瓣，原位缝合，皮瓣下滴注０．２５ｍｌ肝素化生理盐水或ｂＦＧＦ９μｇ。术后７天，计算皮瓣坏死率。再将皮瓣从蒂部开始平均分成近、中、远三段，以定量组织化学方法和生物组织氧耗仪测定ＳＤＨ含量和氧耗量。结果表明，实验组皮瓣坏死率为１８．２１％，对照组为３７．１４％（Ｐ＜０．０５）；实验组中段和远段皮瓣ＳＤＨ含量高于对照组相应部位（Ｐ＜０．０５）；仅有远段皮瓣氧耗量高于对照组相应部位（Ｐ＜０．０５）。提示，ｂＦＧＦ可降低缺血皮瓣坏死率，使皮瓣维持较高的ＳＤＨ含量和氧耗量。其机制可能与ｂＦＧＦ能诱导受创部位毛细血管再生，增加组织血供，减轻皮瓣损伤有关。深入研究ｂＦＧＦ，可能有一定的临床应用价值。     

Effect of spheroid aggregation on susceptibility of primary pig hepatocytes to cryopreservation

Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.     

Characterization of a hollow fiber bioartificial liver device

A three-compartment bioartificial liver (BAL) has been developed for potential treatment of fulminant hepatic failure. It has been shown previously that viability and liver-specific functions were maintained in laboratory-scale bioreactors of such design. In this study, the performance of hepatocytes in a clinical-scale bioartificial liver was verified by sustained specific production rates of albumin and urea, along with oxygen consumption rates for up to 56 h and liver-specific gene expression for up to 72 h. In addition, transmission of porcine endogenous retrovirus and other type C retroviral particles across the hollow fibers was not detected under both normal and extreme operating fluxes. These results demonstrate that the clinical-scale BAL performs at a level similar to the laboratory scale and that it offers a viral barrier against porcine retroviruses.     

Encapsulated multicellular spheroids of rat hepatocytes produce albumin and urea in a spouted bed circulating culture system.

Multicellular spheroids are spherical cell-aggregates that retain tridimensional architecture and tissue-specific functions. For use of multicellular spheroids of hepatocytes in a bioreactor for hybrid artificial liver support, we studied the effect of encapsulation and circulating culture on their integrity and tissue-specific functions. Multicellular spheroids of rat hepatocytes were encapsulated into microdroplets of calcium alginate gel and were used as a bioreactor in medium circulating in a spouted bed chamber. Approximately 10% of the hepatocytes of an adult rat were entrapped in a bioreactor chamber, connected to a gas exchanger and a medium reservoir. The total bed volume of the system was 250 ml. The pH and DO2 of the hormonally defined circulating medium was maintained constantly. Albumin and urea were produced in a linear fashion for 64 h at the rates of 0.02 micrograms/microgram cell protein/day and 0.15-0.2 ng/micrograms cell protein/day, respectively. Viability and structural stability of the spheroids were well preserved after the culture period. These results indicate that these encapsulated multicellular hepatocyte spheroids will provide a useful bioreactor for the continuous production of albumin, in vitro and also a prototype hybrid artificial liver support.     

Efficacy of a Bioreactor Filled with Porcine Hepatocytes Immobilized on Nonwoven Fabric for Ex Vivo Direct Hemoperfusion Treatment of Liver Failure in Pigs

We developed a new bioreactor for a bioartificial liver filled with porcine hepatocytes immobilized on polyester nonwoven fabric (NWF) and in our previous study showed that this NWF bioreactor has promising in vitro efficiency. In the present study, we investigated the efficacy of the NWF bioreactor in a direct hemoperfusion experiment conducted to treat pigs with liver failure. Porcine hepatocytes were isolated from the whole liver of a Sangen strain pig. They were immobilized in a 200 ml column containing NWF via perfusion in a closed circuit for 24 h to prepare a NWF bioreactor. The following day an operative liver failure model was produced by creating a portocaval shunt and ligating the entire hepatoduodenal ligament in the porta hepatis. Perfusion treatment was initiated 4 h after operative induction of liver failure and continued for about 1 h. The pigs which underwent perfusion treatment showed significant improvements in survival and blood data, including ammonia, total bile acid, glucose, and prothrombin time, attributed to significant improvements in the post- as compared to the pre-bioreactor levels in the perfused blood of the treated pigs. These beneficial effects of the NWF bioreactor were based on its excellent composition which allows the accommodation of adequate numbers of hepatocytes and direct contact between hepatocytes and perfused blood.     

Hepatocyte function during experimental use of a bioartificial liver

The aim of the present study was to compare the function of fresh versus cryopreserved hepatocytes in an experimental bioartificial liver system (BAL), especially designed to reproduce clinical parameters. Our BAL consists of a pump, a plasma reservoir, a membrane oxygenator, and a hollow fiber module loaded with 5 x 10(9) isolated porcine hepatocytes, either fresh (n = 5) or cryopreserved (n = 5). In the present setting, the system was isolated and perfused for 6 hours with recirculating plasma obtained from pigs with ischemic liver failure (toxic plasma). The following parameters were studied at 0 and 6 hours: oxygen consumption by the hepatocytes in the bioreactor, hepatocyte viability, as well as plasma concentrations of AST, LDH, ammonia, urea, and total bilirubin. MEGX concentrations were measured following injection of lidocaine into the system 30 minutes after initiation of plasma recirculation. Compared to cryopreserved cells, fresh hepatocytes showed higher viability at both time points studied (P <.05). Furthermore, during BAL sessions, ammonia levels were reduced while urea, AST, and LDH levels were increased with both preservation types (P <.05). Total bilirubin levels increased only during sessions with cryopreserved hepatocytes. After lidocaine administration, both fresh and cryopreserved hepatocytes were capable of producing MEGX; however, fresh-cell bioreactors produced significantly more MEGX at both 30 and 60 minutes after lidocaine administration. Oxygen consumption was significantly higher by fresh-cell bioreactors both before and after BAL use. In conclusion, hepatocytes in the BAL bioreactor showed preservation of important metabolic functions, when perfused with homologous toxic plasma. Fresh cells appeared to respond better than did cryopreserved ones.     

Long-Term Expression of Highly Differentiated Functions by Isolated Porcine Hepatocytes Perfused in a Radial-Flow Bioreactor

To overcome the limitations of standard hollow-fiber module in ensuring efficient cell perfusion and long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which a three-dimensional hepatocyte culture system was perfused in radial-flow geometry. Isolated porcine hepatocytes were cultured for 2 weeks in recirculating serum-free tissue culture medium, in which NH4Cl and lidocaine were repeatedly added, and ammonia removal, urea synthesis, monoethylglycinexylide (MEGX) production, albumin secretion, Po2, Pco2, O2 consumption, and pH were measured thereafter. During the whole duration of the study, ammonia removal was paralleled by urea production, while MEGX concentration was constantly increased. Our results indicated that hepatocytes remained differentiated and metabolically active throughout the duration of the study. The radial-flow bioreactor allowed physiological contact between recirculating fluid and cells by equalizing the concentration of the perfusing components, including O2, throughout the module, suggesting a potential use of this configuration for extracorporeal liver support.     

Significant survival prolongation in pigs with fulminant hepatic failure treated with a novel microgravity-based bioartificial liver

The aim of this study was to evaluate the efficacy and safety of our novel Innsbruck Bioartificial Liver (IBAL; US patent no. 10/641275), which contains aggregates of porcine hepatocytes grown under simulated microgravity, in a porcine model of fulminant hepatic failure (FHF). FHF was induced by a combination of 75-80% liver resection and ischemia of the remnant segments for 60 min in 12 pigs. Two experimental groups were studied: the control group (n = 5) received standard intensive care and the study group (n = 5) received IBAL treatment. The survival of pigs with FHF was significantly prolonged by about 150% with IBAL treatment as compared to controls (controls: 20.4 +/- 2.8 h, IBAL: 51.0 +/- 2.2 h; P = 0.00184). In addition, intracranial pressure, blood ammonia, lactate, aspartate aminotransferase, and alkaline phosphatase levels were lower in the IBAL group than in controls, indicating metabolic activity of porcine hepatocytes in the bioreactor. No adverse effects were observed.     

Extracorporeal bioartificial liver for treating acute liver diseases

Liver is a vital organ of the human body performing myriad of essential functions. Liver-related ailments are often life-threatening and dramatically deteriorate the quality of life of patients. Management of acute liver diseases requires adequate support of various hepatic functions. Thus far, liver transplantation has been proven as the only effective solution for acute liver diseases. However, broader application of liver transplantation is limited by demand for lifelong immunosuppression, shortage of organ donors, relative high morbidity, and high cost. Therefore, research has been focused on attempting to develop alternative support systems to treat liver diseases. Earlier attempts have been made to use nonbiological therapies based on the use of conventional detoxification procedures such as filtration and dialysis. However, the absence of liver cells in such techniques reduced the overall survival rate of the patients and led to inadequate essential liver-specific functions. As a result, there has been growing interest in the development of biological therapy-based extracorporeal liver support systems as a bridge to liver transplantation or to support the ailing liver. A bioartificial liver support is an extracorporeal device through which plasma is circulated over living and functionally active hepatocytes packed in a bioreactor with the aim to aid the diseased liver until it regenerates or until a suitable graft for transplantation is available. This review article gives a brief overview of efficacy of various liver support systems that are currently available. Also, the development of advanced liver support systems, which has been analyzed for improving the important system component such as cell source and other culture and circulation conditions for the maintenance of the liver-specific functions, have been described. Keywords: liver failure, hepatocytes, liver support devices, bioartificial liver, bioreactor.