A bioartificial liver to treat severe acute liver failure.

OBJECTIVE: To test the safety and efficacy of a bioartificial liver support system in patients with severe acute liver failure. SUMMARY BACKGROUND DATA: The authors developed a bioartificial liver using porcine hepatocytes. The system was tested in vitro and shown to have differentiated liver functions (cytochrome P450 activity, synthesis of liver-specific proteins, bilirubin synthesis, and conjugation). When tested in vivo in experimental animals with liver failure, it gave substantial metabolic and hemodynamic support. METHODS: Seven patients with severe acute liver failure received a double lumen catheter in the saphenous vein; blood was removed, plasma was separated and perfused through a cartridge containing 4 to 6 x 10(9) porcine hepatocytes, and plasma and blood cells were reconstituted and reinfused. Each treatment lasted 6 to 7 hours. RESULTS: All patients tolerated the procedure(s) well, with neurologic improvement, decreased intracranial pressure (23.0 +/- 2.3 to 7.8 +/- 1.7 mm Hg; p < 0.005) associated with an increase in cerebral perfusion pressure, decreased plasma ammonia (163.3 +/- 21.3 to 112.2 +/- 9.8 microMoles/L; p < 0.01), and increased encephalopathy index (0.60 +/- 0.17 to 1.24 +/- 0.22; p < 0.03). All patients survived, had a liver transplant, and were discharged from the hospital. CONCLUSIONS: This bioartificial liver is safe and serves as an effective "bridge" to liver transplant in some patients.     

微囊化同系、同种异体、异种肝细胞腹腔移植对急性肝衰的治疗作用

目的 探讨微囊化同系、同种异体、异种肝细胞腹腔移植对急性肝衰（ａｃｕｔｅ ｌｉｖｅｒ ｆａｉｌｕｒｅ,ＡＬＦ）的治疗作用。方法 切除大鼠９０％的肝脏,建立急性肝衰模型;分离纯化包裹同系Ｗｉｓｔａｒ鼠,同种异体ＳＤ鼠及异种豚鼠的肝细胞。移植到受体模型鼠腹腔内,观察微囊存活情况及肝衰鼠的生存率和生化改变。结果 在４８ｈ内对照组存活率仅１５．４％,而同系肝细胞组为７８．６％,ＳＤ微囊组７３．３％,ＧＰ微囊组６２．５％,７ｄ后对照组大鼠全部死亡,而其余３组存活率分别为５７．１％,、５３．３％、５０．０％。微囊化肝细胞组ＡＬＴ和ＴＢＩＬ的水平明显降低,与对照组相比差异有非常显著性（Ｐ〈０．０１）。结论 微囊包裹同种异体和异种肝细胞腹腔移植而不使用免疫抑制剂,能够阻止免疫排斥反应,给予肝功能代谢支持,阻止急性肝衰模型鼠的死     

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.     

Neurological improvement during bioartificial liver sessions in patients with acute liver failure awaiting transplantation

BACKGROUND: Brain edema is the main cause of death in acute liver failure patients awaiting transplantation. We assessed the HepatAssist 2000, a liver-assist system containing porcine hepatocytes, as a bridge to transplantation in patients with acute liver failure. METHODS: Thirteen patients suffering from acute liver failure with criteria for transplantation entered an open baseline-controlled study, with liver-assist treatment sessions at 24-hr intervals until transplantation. Neurological status was regularly evaluated using the Glasgow Coma Scale. RESULTS: Three patients were not treated: one had an immediate transplantation and two improved spontaneously. Ten patients received one to three courses of HepatAssist. A significant neurological improvement (mean Glasgow Coma Scale before and after treatment: 6.5+/-3.7 and 9.6+/-4.4, respectively, P<0.02) was observed, which was related to the volume of plasma processed per square meter of body surface. A significant decrease was observed in mean levels of bilirubin (P=0.0005) and transaminases but not in the other indicators of liver function. Six patients had transient episodes of hemodynamic instability, and five had bleeding complications. Two patients died after transplantation. Eight patients survived with a mean follow-up of 24.3 (18-32) months. CONCLUSION: The HepatAssist 2000 is well tolerated, improves cerebral function, and may be used as a bridge to transplantation for patients with liver failure.     

Transplanted cryopreserved encapsulated porcine hepatocytes are as effective as fresh hepatocytes in preventing death from acute liver failure in rats

BACKGROUND: An implantable bioartificial liver (BAL) using xenogeneic isolated hepatocytes may be an alternative method to orthotopic liver transplantation for treatment of acute liver failure. The purpose of this study was to demonstrate that not only fresh but also cryopreserved porcine hepatocytes could be used in a BAL to prevent death after the onset of acute liver failure in rats. METHODS: Acute liver failure was induced by two-stage 95% hepatectomy. At the time of completion of liver resection, 100 rats were assigned to undergo or not undergo transplantation into the peritoneum of 4 meters of hollow fibers filled with 60 million either fresh or cryopreserved porcine hepatocytes, or syngeneic hepatocytes, or culture medium, or of 60 million nonencapsulated cryopreserved porcine hepatocytes without immunosuppressive therapy. Survival rates at 7 days were compared between the different groups. RESULTS: In the control groups of hepatectomized animals not receiving encapsulated hepatocytes, 69-79% of the rats died from acute liver failure. The mortality rate was reduced to 15% (2 of 13) in rats receiving fresh porcine hepatocytes (P<0.01), 25% (4 of 16) in rats transplanted with either cryopreserved or syngeneic hepatocytes (P<0.05). Survival rates were maintained when hollow fibers were explanted > or =4 days after hepatectomy. In surviving rats, the weight of the remnant native liver increased with time and returned to the initial weight after 1 month. CONCLUSIONS: The implantable BAL using xenogeneic porcine hepatocytes was able in preventing death from acute liver failure without immunosuppressive therapy. Encapsulated cryopreserved hepatocytes were as effective as fresh hepatocytes.     

新型多层平板型生物人工肝治疗急性肝功能衰竭犬的疗效

目的 评价新型多层平板型生物人工肝治疗急性肝功能衰竭动物的疗效.方法 以新鲜猪肝细胞及猪骨髓基质干细胞为细胞来源,共培养于新型多层平板型生物反应器内,从而构建一种新型的生物人工肝.采用D-氨基半乳糖给药方式构建犬急性肝功能衰竭模型,实验组(n=8)给予生物人工肝治疗;对照组(n=8)仅给予一般监护.观察和检测所有动物一般情况、生化指标及生存率.结果 实验组动物经生物人工肝治疗后,肝性脑病及一般精神状况均得到较明显改善,丙氨酸氨基转移酶从( 1512±183) U/L降至(86±25) U/L;天冬氨酸氨基转移酶从(1472±365) U/L降至(46±1l)U/L;乳酸脱氢酶从(463±76) U/L降至(312±84)U/L;总胆红素从(28.8±6.2) μmol/L降至(12.5±3.6) μmol/L;血氨从(56±15)μmol/L降至(34±10) μmol/L,同时凝血功能及白蛋白水平亦得到改善.8条犬中,5条存活、3条死亡,治疗过程中未出现严重并发症.对照组动物一般情况未见明显改善,各项化验指标呈逐渐加重趋势,最终8条犬中5条死亡,3条存活.但两组生存率的差异无统计学意义(P=0.294).结论 新型多层平板型生物人工肝对急性肝功能衰竭动物具有显著疗效,是治疗急性肝功能衰竭的一种有效支持手段.     

Fulminant liver failure: clinical and experimental study.

Clinical experience of some newer methods of hepatic support is described. The results are unpredictable and far from satisfactory. The need for an animal model in which potential therapeutic methods can be studied is emphasized. Such a model based on carefully imposed ischaemic insult to the liver in the absence of portacaval shunting is described. It is suggested that bacterial presence in the bowel together with a depression of the liver reticuloendothelial function plays an important part in the early and rapid mortality of acute liver failure. Temporary auxiliary liver transplantation using an allograft or a closely related primate heterograft seem to be the 2 best available methods of hepatic support for potentially reversible acute liver failure.     

Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes

Abstract: Background: The aim of this study was to evaluate in vitro and in vivo functions of isolated hepatocytes after immortalization, cryopreservation, encapsulation and xenotransplantation into mice with fulminant liver failure (FLF). Methods: Rat and human hepatocytes were isolated from normal liver tissue by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors coding for SV 40 large T antigen, Bmi-1 and telomerase. Rat and immortalized human hepatocytes (IHH) were encapsulated in 400 micron alginate-PLL-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or freshly isolated) were cultured in albumin-free medium and albumin production was measured by enzyme-linked immunosorbent assay (ELISA). In vivo, a model of FLF was established in C57/BL6 mice by acetaminophen administration (700 mg/kg i.p.) followed 15 h later by a 30% hepatectomy. Microencapsulated (cryopreserved or freshly isolated) hepatocytes were transplanted intraperitoneally to mice with FLF and the following experimental groups were performed: group 1 (n = 10) Tx of empty capsules; group 2 (n = 12) Tx of free primary rat hepatocytes; group 3 (n = 12) Tx of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) Tx of fresh encapsulated rat hepatocytes; group 5 (n = 9) Tx of cryopreserved encapsulated IHH; group 6 (n = 10) Tx of fresh encapsulated IHH. Animals were killed at regular intervals and histopathology of microcapsules and liver tissue was obtained. Results: In vitro, cryopreserved or fresh encapsulated rodent hepatocytes showed a progressively decreasing albumin secretion over 1 week in culture. In contrast, cryopreserved or fresh encapsulated IHH showed minimal, but stable albumin secretion. In vivo, FLF was achieved by combination of acetaminophen with 30% hepatectomy, resulting in a reproducible survival of 23% ± 5%. In groups 1 and 2, survival rates were not improved significantly compared with untreated mice. In groups 3 and 4, Tx of cryopreserved or fresh encapsulated rat hepatocytes significantly increased survival rate to 66% and 80%, respectively (P < 0.01). In groups 5 and 6, Tx of cryopreserved or fresh encapsulated IHH improved survival to 50% and 55%, respectively (P < 0.05). Histopathology revealed that encapsulated hepatocytes were viable up to 2 weeks post-Tx. Conclusions: Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation short-term. IHH showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes loose specific functions after immortalization. After induction of FLF in mice, intraperitoneal Tx of encapsulated (primary or immortalized, fresh or cryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naïve and genetically modified hepatocytes can successfully be encapsulated, stored using cryopreservation, and be transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.     

Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes

BACKGROUND: The aim of this study was to evaluate the in vitro and in vivo function of hepatocytes after immortalization, cryopreservation, encapsulation, and xenotransplantation into mice with fulminant liver failure (FLF). METHODS: Rat and human hepatocytes were isolated by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors. Rat and immortalized human hepatocytes (IHH) were encapsulated in 400 microm of alginate-poly-L-lysine (PLL; Sigma, Buchs, Switzerland)-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or noncryopreserved) were cultured; albumin secretion was measured by enzyme-linked immunosorbent assay. Microencapsulated (cryopreserved or noncryopreserved) hepatocytes were transplanted intraperitoneally to mice with FLF: group 1 (n = 10) transplantation of empty capsules; group 2 (n = 12) transplantation of free primary rat hepatocytes; group 3 (n = 12) transplantation of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) transplantation of encapsulated rat hepatocytes; group 5 (n = 9) transplantation of cryopreserved encapsulated IHH; group 6 (n = 10) transplantation of encapsulated IHH. RESULTS: Compared with free primary hepatocytes, cryopreserved or noncryopreserved encapsulated rodent hepatocytes showed similar levels of continuous in vitro albumin secretion over 1 week. Cryopreserved or noncryopreserved encapsulated IHH showed minimal albumin secretion compared with free primary human hepatocytes. Fulminant liver failure, produced by a combination of acetaminophen and 30% hepatectomy, resulted in a 20% to 30% host survival. In groups 1 and 2, survival was unmodified, compared with untreated mice. For groups 3 and 4, transplantation of cryopreserved or noncryopreserved encapsulated rat hepatocytes significantly increased survival rates to 66% and 80%, respectively (P < .01). For groups 5 and 6, transplantation of cryopreserved or noncryopreserved encapsulated IHH improved host survival to 50% and 55%, respectively (P < .05). CONCLUSIONS: Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation. Immortalized human hepatocytes showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes lose some specific functions after immortalization. After induction of FLF in mice, intraperitoneal transplantation of encapsulated (primary or immortalized, cryopreserved or noncryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naive and genetically modified hepatocytes can be successfully encapsulated, stored by cryopreservation, and transplanted into xenogeneic recipients with FLF to sustain liver metabolic functions.     

生物人工肝构建及临床应用14例次报告

目的　研究用聚砜膜纤维管构建的新型生物人工肝是否能有效支持肝脏功能。方法应用两步胶原酶法分离猪肝细胞 ,构建聚砜膜中空纤维管生物反应器 ,细胞数量 1× 10 10 个 ,与非生物人工肝同期或非同期使用 ,对 12例患者治疗 14例次 ,每次支持时间为 6h ,治疗前后观察患者一般状况并检测血氨、凝血酶原时间和部分肝功能指标。结果　应用生物人工肝治疗后血氨、凝血酶原时间和总胆红素均明显改善 ,治疗 2d后血氨仍为 (10 6± 131) μmol/L ,与治疗前相比较 [(172± 187)μmol/L]差异有显著意义 (P <0 0 5 ) ;治疗后 1个月 ,同期生物人工肝治疗组死亡 1例 ,非同期生物人工肝治疗组死亡 2例 ,患者总存活率 75 % (9/12 )。结论　我们构建的新型生物人工肝可支持急性肝功能衰竭患者的肝功能 ,同期生物人工肝治疗可能优于非同期生物人工肝。     

异种肝细胞移植防治大鼠急性肝功能衰竭

目的: 探讨异种肝细胞移植对大鼠急性肝功能衰竭的治疗效果. 方法: 切除大鼠90%肝脏,建立急性肝功能衰竭模型.分离异种豚鼠和同种异体Sprague-Dawley鼠肝细胞,切肝术前1天植入受体脾脏内.观察受试大鼠存活时间、切肝术后24小时血生化改变和脾脏切片表现. 结果: ①中位存活时间,对照组为21小时,同种组为56小时,异种组为40小时.同种组存活时间较对照组长(P<0.01),异种组亦延长(P<0.05).②移植组部分血生化指标有所改善.异种组中仅血糖和凝血酶原时间改善较明显(P<0.05),同种组中谷丙转氨酶、总胆红素、血糖和凝血酶原时间都有明显改善(P<0.05或P<0.01).③肝细胞植入后48小时,异种组脾脏内仅存少量萎缩的肝细胞,同种组脾脏内仍散在健存肝细胞. 结论: 异种肝细胞移植对大鼠急性肝功能衰竭有防治作用;要提高移植效果,首先需克服早期的免疫排斥.     

Hepatocyte transplantation in the treatment of acute liver failure: microencapsulated hepatocytes versus hepatocytes attached to an autologous biomatrix

A liver transplant is considered today to be the only effective therapeutic solution for many otherwise intractable hepatic disorders. However, liver transplantation is beset by shortage of donors. Over the years, many liver support systems have been developed to supply the liver functions, mostly as a bridge to transplantation. Transplantation of isolated hepatocytes (HcTx) instead of whole liver has constituted one of the most appealing possibilities to treat several diseases. We compared two different models of HcTx in a surgical model of acute liver failure in pigs, using microencapsulated hepatocytes (MHcTx) and hepatocytes attached to a porcine biomatrix (PBMHcTx), both transplanted into peritoneum. The collected data were survival, laboratory findings, hemodynamic parameters, light microscopy, histology, MTT, and glycogen content. The group with PBMHcTx has a better outcome than the group with MHcTx (p < 0.05). Histology showed normal morphology of the hepatocytes, high glycogen content, 75% viability, positive MTT, and 95% adhesion of the hepatocytes to the biomatrix. Our biomatrix (PBM) provides cell-to-cell contact and interaction with extracellular matrix, which have been shown to play major roles in hepatocyte survival and physiologic regulation of gene expression, and guarantee a prompt engraftment and an adequate neovascularization. PBMHcTx is a useful method to treat acute liver failure and it indicates a possible liver-direct gene therapy in the treatment of inherited and acquired disorders.     

Dormant liver metastases: an experimental study.

Experimental work was undertaken to evaluate whether intrahepatic recurrences, observed after resection of colorectal liver metastases in humans, could be due to the activation of dormant cancer cells already present within the liver at liver resection. About 250 cell aggregates (DHDK12 colon carcinoma cell line) were injected into the portal vein of 70 BD IX rats. Eight weeks later, 43 rats with no apparent liver metastases were divided randomly into three groups: group 1 (n = 15) served as control; group 2 (n = 15) were given cyclosporin A (10 mg kg body-weight-1 day-1) for 28 days; and group 3 (n = 13) underwent a 70 per cent hepatectomy. Twelve weeks after the injection of cells, when the animals were killed, 20 per cent of rats in group 1 had liver metastases, 80 per cent in group 2 (P less than 0.01) and 62 per cent in group 3 (P less than 0.05). Undetectable liver micrometastases may have been present at 8 weeks and had not developed until stimulation by cyclosporin A-induced immunosuppression or by liver regeneration after hepatectomy. A similar mechanism may occur clinically and explain some of the recurrences observed after resection of liver metastases.     

Mild hypothermia prevents cerebral edema in acute liver failure

Mild hypothermia prevents the development of brain edema in rats with acute liver failure resulting from hepatic devascularization. Mechanistic studies performed in this model suggest that the protective effect of hypothermia results from the inhibition of blood-brain transfer of ammonia, an action which could result (at least in part) from an effect on cerebral blood flow. Hypothermia-induced reductions of brain ammonia are associated with normalization of extracellular brain glutamate concentrations in rats with acute liver failure. Studies in humans suggest that mild hypothermia is beneficial in the management of severely raised intracranial pressure, both before and after liver transplantation in patients with acute liver failure due to acetaminophen overdose. Mild hypothermia offers a potentially useful bridge therapy in patients with acute liver failure who are awaiting liver transplantation. Received: July 7, 2000 / Accepted: October 12, 2000