Measurement and Destruction of Porcine Endogenous Retrovirus in the Chinese Bama Minipig

Porcine hepatocytes are widely used in bioartificial liver (BAL) systems for the treatment of liver failure, and Chinese Bama minipigs (BMPs) are extensively used for animal experiments in the field of medicine in China. The genome of porcine endogenous retroviruses (PERVs) has not yet been accurately quantified, posing a threat to their clinical application because they act as a source of cells. In this study, we used genome sequence data from BMPs to predict PERV copies and their distribution. We validated and quantified the identified PERV copies and subtypes across different BMP individuals and tissues using quantitative real-time polymerase chain reaction and droplet digital polymerase chain reaction, respectively, and found that the BMP genome contains only 11 to 21 PERV copies. Notably, they lack the C subtype, which is a relatively safe cell source. Moreover, we applied CRISPR/Cas9 technology to knock out the pol fragment of PERVs in primary renal fibroblasts (PRFs) from BMPs and obtain PERV-destructed cells. Overall, our results lay a foundation for obtaining PERV-destructed BMPs as a safe source of hepatocytes for BALs for future applications.     

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver

Di Nicuolo G, D’Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RAFM. Long‐term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell–based Academic Medical Center bioartificial liver. Xenotransplantation 2010; 17: 431–439. © 2010 John Wiley & Sons A/S. Abstract: Background: Clinical use of porcine cell–based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow‐up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. Methods: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center‐BAL (AMC‐BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real‐time polymerase chain reaction assay has been used. Results: Eight patients who received a liver transplant after AMC‐BAL treatment are still alive under long‐term pharmacological immunosuppression. The current clinical follow‐up ranges from 5.6 to 8.7 yr after BAL treatment. A new q‐real‐time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥5 copies per 1 × 10⁵ PBMCs. The linear dynamic range was from 5 × 10⁰ to 5 × 10⁶ copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. Conclusion: Up to 8.7 yr after exposure to treatment with porcine liver cell–based BAL, no PERV infection has been found in long‐term immunosuppressed patients and in HCWs by a new highly sensitive and specific q‐real‐time PCR assay.     

Efficacy of an extracorporeal flat-plate bioartificial liver in treating fulminant hepatic failure

BACKGROUND: Fulminant hepatic failure is associated with a high mortality rate. Orthotopic liver transplantation is the only established treatment for patients who do not respond to medical management. A major limitation of this treatment is a shortage of donor organs, resulting in many patients dying while waiting for a transplant. An extracorporeal bioartificial liver (BAL) has the potential to provide temporary support for patients with fulminant hepatic failure (FHF) and for patients awaiting orthotopic liver transplantation. We developed a flat-plate BAL with an internal membrane oxygenator in which porcine hepatocytes were cultured as a monolayer. MATERIALS AND METHODS: Twenty-four hours after cannulation of the left carotid artery and right jugular vein, FHF was induced in rats by administering 2 intraperitoneal injections of D-galactosamine (GalN) (1.2 g/kg) at a 12-h interval. The rats were connected to a BAL device 24 h after the first GalN injection and underwent extracorporeal perfusion for a duration of 10 h. Liver histology, liver-specific markers, and animal survival up to 168 h (7 days) were examined. RESULTS: Histologically, liver damage was reduced in the animal group treated with the hepatocyte-based BAL device. Significant reductions occurred in the plasma ammonia levels and prothrombin times in the group treated with the seeded BAL device. Animal survival in the group treated with the seeded BAL device was significantly higher (50.0%) than in the control animal group treated with an unseeded BAL device (11.1%). CONCLUSIONS: This flat-plate BAL with an internal membrane oxygenator and cultured porcine hepatocytes has yielded encouraging results in the treatment of rats with GalN-induced FHF.     

Internal bioartificial liver with xenogeneic hepatocytes prevents death from acute liver failure: an experimental study.

OBJECTIVE: To demonstrate that a bioartificial liver, using allogeneic or xenogeneic hepatocytes protected from rejection by a semipermeable membrane, could prevent death from acute liver failure. SUMMARY BACKGROUND DATA: An implantable bioartificial liver using isolated hepatocytes could be an alternative to orthotopic liver transplantation to treat patients with acute liver failure. It could serve either as a bridge until liver transplantation or as the main treatment until recovery of the native liver. However, allogeneic or xenogeneic hepatocytes that could be used in clinical applications are spontaneously rejected. METHODS: Acute liver failure was induced in rats by 95% liver resection. Twenty-five million hepatocytes harvested in rats (allogeneic) or guinea pigs (xenogeneic) were encapsulated in a semipermeable membrane to protect them from rejection. The hollow fibers containing hepatocytes were transplanted into the peritoneum of recipient rats. Survival rates were compared between rats transplanted or not with hepatocytes. RESULTS: In groups not transplanted with viable hepatocytes, 73% to 93% of rats died after 95% liver resection. The mortality rate was reduced to 39% in rats transplanted with allogeneic hepatocytes and 36% in rats transplanted with xenogeneic hepatocytes. The bioartificial liver could be removed 1 month after transplantation, when regeneration of the native liver was complete. Allogeneic and xenogeneic hepatocytes remained viable. CONCLUSIONS: The implantable bioartificial liver was able to prevent death in this model of acute liver failure. This could be an important step toward clinical application of the method.     

In vitro and in vivo evaluation of albumin synthesis rate of porcine hepatocytes in a flat-plate bioreactor

Several configurations of extracorporeal bioartificial liver devices have been developed for the potential treatment of fulminant hepatic failure or as a bridge to liver transplantation. Recently, we developed a microchannel flat-plate bioreactor with an internal membrane oxygenator in which porcine hepatocytes are cultured as a monolayer on the bottom glass surface. In the present study, we investigated synthetic function of porcine hepatocytes in the bioreactor in both in vitro and in vivo flow circuit models. In vitro, albumin synthesis was stable in the bioreactor for up to 4 days of perfusion. In vivo, with the extracorporeal connection of the bioreactor to rat vasculature, porcine albumin was detectable for 24 h in the rat plasma. We also developed a simple mathematical model to predict the in vivo porcine albumin concentration in rat plasma. These results indicate that this configuration of a microchannel flat-plate bioreactor has potential as a liver support device and warrants further investigation.     

Bridging a patient with acute liver failure to liver transplantation by the AMC-bioartificial liver

Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 x 10(9) viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.     

Safe and efficient gene transfer into porcine hepatocytes using Sendai virus-cationic liposomes for bioartificial liver support

Establishment of a bioartificial liver support system using genetically modified hepatocytes is a potential approach to improve the treatment of severe liver failure. We describe the development of an efficient ex vivo method of gene transfer into a large number of porcine hepatocytes using hemagglutinating virus of Japan (HVJ)-liposome. The transfection efficiency of HVJ-liposome into isolated porcine hepatocytes attached to microcarrier beads was evaluated by beta-galactosidase (beta-gal) staining, fluorescence activated cell sorting analysis for beta-gal and luciferase assay, respectively. To examine the function and cellular damage of transduced hepatocytes, we used enzyme-linked immunosorbent assay for porcine albumin synthesis, lidocaine clearance test (P-450 activity), aspartate aminotransferase, and lactic dehydrogenase release assays. The optimal conditions for gene transfer into the beads-attached hepatocytes using HVJ-liposome included 4 microg of deoxyribonucleic acid with 200 microg of lipid/2 x 105 cells and exposure duration of 90 min. Under these conditions, beta-gal and luciferase genes were transduced to 2.5 x 108 isolated porcine hepatocytes following attachment to the beads. Positive beta-gal staining was observed in more than 30% of the beads-attached hepatocytes. The gene transfer activity of HVJ-liposome method determined by luciferase activities was about 100-fold of that of the lipofection method. Transfected porcine hepatocytes remained functional without any significant cell damage. Our results demonstrated that HVJ-liposome mediated gene transfer into microcarrier-attached porcine hepatocytes is an efficient and nontoxic method suitable for a bioartificial liver support sytem.     

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.     

猪肝细胞与骨髓间充质干细胞最适共培养体系的建立

目的：建立猪肝细胞与骨髓间充质干细胞（MSCS）最适宜共培养体系,为生物人工肝的构建提供理想的细胞来源。方法：抽取中华实验猪（n=3）髂前上棘骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;采用原位两步胶原酶法分离猪肝细胞后与MSCS按1∶1,2∶1,5∶1和10∶1的比例混合培养,观察各组共培养肝细胞形态和功能的变化水平。结果：所获肝细胞纯度>99%,存活率>95%。共培养组肝细胞迅速黏附于MSCS表面,在三维空间呈球形聚集生长;肝细胞与MSCS间出现细胞连接,超微结构与正常肝细胞接近。肝细胞/MSCS 以2∶1共培养组的肝细胞清蛋白分泌水平、尿素合成能力和细胞色素P450为各组中之最佳,自第1天培养起均显著高于对照组（P<0.05）,并在第2天达到高峰,而且下降趋势较缓慢。结论：猪肝细胞/MSCS 按2∶1组成的最适共培养体系可最大程度地维持肝细胞功能,为构建功能性生物人工肝提供高效细胞材料。     

AN69 hollow fiber membrane will reduce but not abolish the risk of transmission of porcine endogenous retroviruses

As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supematant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.     

Membrane barrier of a porcine hepatocyte bioartificial liver

Pores in the membrane of a bioartificial liver (BAL) allow it to function as a semipermeable barrier between its contents (i.e., liver cells) and components of the recipient's immune system. This study is designed to assess the influence of pore size on immune response to a BAL containing porcine hepatocytes. Sixteen healthy dogs were divided into four groups (four dogs per group) based on pore size of the BAL membrane and level of exposure to porcine hepatocytes. Group 1 dogs were administered porcine hepatocytes by intraperitoneal injection and served as positive controls. Group 2 dogs were exposed to porcine hepatocytes in a large-pore (200-nm) BAL, and group 3 dogs were exposed to porcine hepatocytes in a small-pore (10-nm) BAL. Group 4 dogs were exposed to a no-cell (unloaded) BAL and served as negative controls. Intraperitoneal injection of hepatocytes or 3 hours of BAL hemoperfusion was performed day 0 and 3 weeks later on day 21. Biochemical, humoral, and cellular measures of immune response were collected until day 44. The initiation of BAL hemoperfusion was associated with a rapid decline in CH₅₀ levels of complement and transient neutropenia and thrombocytopenia during all BAL exposures. Xenoreactive antibody response to BAL was increased by use of membranes with large pores and secondary exposures. Skin testing on day 42 showed a delayed-type hypersensitivity response to porcine hepatocytes that also correlated with level of previous antigen exposure. BAL treatment was associated with both immediate and elicited immunologic responses. The immediate response was transient and not influenced by membrane pore size, whereas elicited responses were influenced by pore size of the BAL during previous exposures. (Liver Transpl 2003;9:298-305.)     

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.     

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.     

Natural antibodies prevent in vivo transmission of porcine islet-derived endogenous retrovirus to human cells

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.