Author Affiliations: Department of Hepatobiliary Surgery (Han B, Shi XL, Yuan XW, Ren HZ and Ding YT) and Department of Osteology (Qiu Y), the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China; Department of General Surgery, the Third Affiliated Hospital of Suzhou University, Changzhou 213000, China (Zhang Y); and State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210008, China (Gu ZZ)

Corresponding Author: Yi-Tao Ding, Professor, Department of Hepatobiliary Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing 210008, China (Tel: +86-25-83304616ext66866; Fax: +86-25-83317016; Email: yitaoding@hotmail.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60401-5

Published online July 15, 2015.

 

 

Contributors: DYT proposed the study. HB, SXL, ZY, YXW and RHZ performed research and wrote the first draft. GZZ and QY gave the technical guidance. HB collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. DYT is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81300338), Postdoctoral Fellowship of Jiangsu province (1202057C) and Project funding of Clinical Medical Center of Digestive Disease in Jiangsu province (BL2012001).

Ethical approval: All animal procedures were performed according to the institutional and national guidelines approved by the Animal Care Ethics Committee of Nanjing University and Nanjing Drum Tower Hospital.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL.

 

METHODS: Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity.

 

RESULTS: PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative.

 

CONCLUSION: No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

 

(Hepatobiliary Pancreat Dis Int 2015;14:492-501)

 

KEY WORDS: porcine endogenous retrovirus; hybrid bioartificial liver; porcine hepatocyte; acute liver failure

Liver transplantation is the only approved effective treatment for patients with acute liver failure (ALF). However, the shortage of liver donors is still the major cause of high mortality rate in patients who are waiting for liver transplantation. In the last two decades, the expanding gap between the number of patients on the waiting list for liver transplantation and the number of available liver donors has highlighted the strong need for a temporary liver support system.^[1] Therefore, extracorporeal liver-assist devices have been developed to bridge these patients to transplantation or to allow the native liver to regenerate. The ideal liver support devices should provide the primary functions such as detoxification, biosynthesis and regulation. Hybrid bioartificial livers (HBALs) can integrate biological functions of hepatocytes with effective detoxification function of non-bioartificial liver.

 

In this study, a novel HBAL with the mimic liver microenvironment was developed on the basis of an anionic resin adsorption column and a multi-layer flat-plate bioreactor carrying co-cultured porcine hepatocytes and bone marrow mesenchymal stem cells (MSCs).^{[2, 3]} Like other porcine bioartificial livers (BALs), the safety issue regarding porcine endogenous retrovirus (PERV) transmission cannot be ignored because a large number of human cell infection in vitro and rodent animal infection in vivo have been reported.^[4] Previously, the microbiological safety of a simple BAL built in our institute was studied preliminarily by treating healthy dogs.^[5] In this preclinical experiment, eight canines with D-galactosamine-induced ALF were treated by a novel HBAL and the microbiological safety of this novel HBAL was evaluated by determining the PERV transmission.

 

 

Eight outbred white pigs with an average body weight of 15-20 kg and eight Chinese field dogs with an average body weight of 11-13 kg received humane care, and all animal procedures were performed according to the institutional and national guidelines approved by the Animal Care Ethics Committee of Nanjing University and Nanjing Drum Tower Hospital. All cell culture-related reagents were purchased from GIBCO, USA.

 

Dogs under general anesthesia were administered with D-galactosamine (Sigma, USA) dissolved in 5% glucose solution at a dose of 1.5 g/kg through the ulnar vein.^[6] Venous blood samples were collected every other day for biochemical analysis until the 5th day after HBAL treatment.

 

The co-culture system of porcine hepatocytes and MSCs was prepared according to previous reports.^[3] In brief, porcine MSCs were collected by gradient centrifugation (20 minutes, 400 g, density 1.077 g/mL) and seeded at a density of 1×10⁶ cells/cm² in growth medium containing low-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 100 mg/mL streptomycin. The cultured MSCs were confirmed by surface markers such as CD45, CD29, CD44 and CD90 via FACScan (Becton Dickinson, San Jose, CA, USA). Primary porcine hepatocytes were then harvested by a two-step in situ collagenase perfusion technique. The viability of the isolated primary hepatocytes determined by trypan blue exclusion was more than 95%. Nonparenchymal cells, as judged by their size (<10 µm in diameter) and morphology (nonpolygonal or stellate), were verified by immunocytochemical analysis of albumin and cytokeratin 18 with the population of less than 1%. The mixed suspension of fresh hepatocytes and MSCs during the passages of 3-5 at the ratio of 2:1 was perfused at a density of 1×10⁶ cells/mL in the substratum of 500 mL RPMI-1640 medium without serum and incubated in our new bioreactor supplemented with 5% CO₂ at 37 ��.

 

The novel HBAL was composed of three roller pumps, a heparin pump, an infusion heater, a plasma filter (Sorin Group, Mirandola, Italy), an anionic resin adsorption column (Aier, China), a plasma component separator (Kawasumi Laboratories, Tokyo, Japan) serving as immunoprotective barrier, an oxygenation device and a multi-layer radial-flow bioreactor with galactosylated chitosan nanofiber scaffolds. The bioreactor and the oxygenation device were prepared and kept in an incubator with the internal temperature of 37 �� as previously reported.^[2] The whole system was then assembled according to Fig. 1.

 

The ALF canines were catheterized via the internal carotid artery and internal jugular vein under continuous anesthesia by intravenous administration of diprivan at a dose of 10 mg/kg per hour and then connected to the HBAL device according to Fig. 1, on the first day after the establishment of the model. HBAL treatment was initiated after the cells were seeded for 4 hours and most cells were adhered to the galactosylated chitosan nanofiber scaffolds. The whole blood in the first circuit was perfused at a rate of 40 mL/min for 6 hours, the plasma was separated from the plasma filter at a rate of 15 mL/min in the second circuit, and the plasma passed through the plasma component separator at a rate of 15 mL/min in the third circuit. Heparin was administered intravenously at a dose of 100 U/kg at the beginning of treatment, reduced to a dose of 40 U/kg per hour in the first circuit, and finally removed at 30 minutes before the end of the treatment. The plasma circulating in the HBAL and the whole blood in the dogs were collected at regular intervals until the end of the experiment after one year treatment followed by the sacrifice of the dogs and the retrieval of tissues such as the heart, liver, spleen, lung and kidney. The peripheral blood mononuclear cells (PBMCs) and plasma were separated from the whole blood, and the tissues were preserved at -80 �� for future use.

 

Total RNA was extracted from PBMCs, plasma and tissues with Trizol (Invitrogen, USA) and then treated with DNase I (Invitrogen, USA) according to the manufacturer's instructions. The extracted RNA with OD_{260/280 in the range of 1.60-2.00 was reversely transcribed to cDNA using reverse transcriptase (RT) kits (Biouniquer, China) in accordance with the instructions. Total DNA was extracted from the plasma, separated PBMCs and tissues using DNA extracting kits (Axygen, USA) according to the manufacturer's instructions. PCR was completed using the specific primers (Table 1).}^{[7, 8]} The amplified products were evaluated by 2% agarose gel electrophoresis with ethidium bromide staining.

 

RT activity in serum and tissue homogenate was detected by C-type RT kits (Cavidi-Tech, Uppsala, Sweden) according to the manufacturer's instructions.

 

In vitro infection experiments were conducted according to the previous report with minor modification.^[9] HEK293 cells (as the gift from Professor Zi-Chun Hua at Nanjing University) were passaged overnight in 25-mL tissue culture flasks and then incubated in the culture composed of 2 mL of separated plasma at definite time and 3 mL of high-glucose Dulbecco's modified Eagle's medium with 0.8 g/mL polybrene (Sigma-Aldrich, USA). Meanwhile, the supernatant of PK15 cells and 0.8 g/mL polybrene was used to infect HEK293 cells as the positive control. After 4 hours exposure at 37 ��, the inoculum was removed and the monolayer cells were washed with phosphate buffered solution (PBS) twice. Then, the cells were cultured in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum until the appropriate confluence for collection. The harvested HEK293 cells were used to test PERV DNA by PCR. RT activity was determined in the supernatant of the HEK293 cells.

 

Western blotting was used to detect PERV capsid protein in tissue samples. Fifty micrograms of the extracted protein from tissue samples were subjected to the separation in 12.5% denatured SDS-PAGE and then transferred to polyvinylidene difluoride membrane by electroblotting. The membrane was blocked using 0.1% Tween-20 and 5% skimmed milk in PBS. The mouse anti-FeLV p27 antibody (Abcam, USA) that has cross-reaction with capsid protein gag p30 of PERV^[10] was incubated with the membrane at the dilution ratio of 1:1500 overnight at 4 ��, and then followed by the incubation with goat anti-mouse IgG conjugated horseradish peroxidase at the dilution ratio of 1:10?000 (KeyGEN, Nanjing, China) for one hour at 37 ��. Immunoreactive proteins on the membrane were detected with an enhanced chemiluminescence system with the exposure time of 15-20 seconds on hyperfilm ECL (Kodak, USA). Meanwhile, β-actin was used for loading control, and the lysate of PERV-infected HEK293 cells was determined as the control.

 

The immunohistochemical detection of PERV in tissues was conducted according to the previous reports.^[7] In brief, the tissue samples were snap-frozen in pre-chilled isopentane and the section with the thickness of 4 µm was fixed with acetone and washed with PBS. A standard two-step indirect staining was conducted by incubating with mouse anti-FeLV p27 antibody as the primary antibody at a dilution ratio of 1:200 overnight at 4 �� and goat anti-mouse horseradish peroxidase-labeled antibody (KeyGEN, Nanjing, China) as the secondary antibody at a dilution ratio of 1:1000 at 37 �� for 30 minutes. Porcine tissues were used as the positive controls.

 

 

The heart rate and respiratory rhythm of each animal with continuous recording by an electrocardiogram monitor were stable during exogenous perfusion. Seven out of 8 dogs could stand and intake food within 7 days after the end of the treatment, and 7 dogs survived during the entire study for one year and one dog died at the 6th day after the treatment. Several laboratory parameters including ALT, AST, LDH, total bilirubin, albumin, ammonia, and PT were partially improved (Table 2).

 

Agarose gel electrophoresis exhibited no PERV DNA or RNA in PBMCs harvested at the various time points (Fig. 2). Moreover, the screening for porcine-specific gene revealed no positive Sus scrofa cytochrome B (SsCytB) in the DNA of all canine PBMCs.

 

During the screening of the plasma, all RT-PCRs of the RNA from the collected plasma were negative except the positive bands of protease and polymerase in the 3rd circuit (Fig. 3).

 

RT activity was examined twice for all collected plasma samples and limited in the plasma in the 3rd circuit before the treatment and at the 3rd hour during the treatment. RT activity was not detected in other samples including dog plasma collected during and after the treatment (Table 3).

 

In in vitro infection experiments, PERV-specific genes including protease and polymerase genes as well as the porcine-specific SsCytB gene were simultaneously observed in the DNA from HEK293 cells incubated with the plasma from the 3rd circuit. The optical density of the bands was analyzed by software ImageJ to reveal obviously lower PERV-specific genes when compared with the SsCytB gene, suggesting that PERV-specific genes resulted from microchimerism rather than infection. The DNA assay of other HEK293 cells incubated with the plasma from the canines or other circuits was negative. In addition, negative RT activity was observed in all supernatant from HEK293 cells, which was another confirmation of non-infection.

 

On electrophoretic images (Fig. 4), no PERV DNA and RNA as well as porcine-specific SsCytB genes were observed in the retrieved tissue samples such as heart, liver, spleen, lung, and kidney.

 

Western blotting showed no PERV capsid protein gag p30 in tissue lysates of these organs (Fig. 5). Additionally, during the immunohistochemical detection of the tissue section, no cell with positive PERV-specific protein in each tissue was observed (Fig. 6), while RT activity was detected in the tissue lysates.

 

 

Because the clinical trials of other BALs are not very optimistic,^[1] we developed a novel HBAL which was based on an anionic resin adsorption column and a multi-layer flat-plate bioreactor carrying co-cultured porcine hepatocytes and bone marrow MSCs. The bioreactor is composed of a stack of 65-layer round flat-plates and a cylindrical container with an inlet on the top and an outlet on the bottom. In order to improve the cellular functions, each plate is covered with nanofiber scaffolds to mimic the topography of extracellular matrix and the galactose is grafted onto the nanofibers to mimic the biochemical environment of extracellular matrix.^{[2, 11]} Moreover, the ratio of porcine hepatocytes and MSCs in the co-culture system was 2:1. The previous studies^{[3, 12]} showed that this ratio is optimal for the function of the cultured cells.

 

However, as xenogeneic cells, porcine HBAL still has some problems such as microbiological safety. PERV was first discovered in 1971 in PK15 cells.^[13] In 1997, Patience et al^[14] found that PERV released from PK15 infected HEK293 cells in vitro, suggesting that PERV has the capability to infect a variety of human cells such as endothelial cells, fibroblasts and bone marrow stromal cells in vitro with fast virus replication.^[4] A study^[15] on cross-species PERV infectivity in vivo showed that PERV infects guinea pigs but the virus does not replicate. However, the transmitted PERV has the capability to replicate in mice with nonobese diabetic severe combined immunodeficiency.^{[16, 17]} These investigations suggested that PERV transmission as a microbiological safety issue cannot be ignored during the application of BAL/HBAL carrying porcine hepatocytes, especially when the recipients are under immunosuppression status.^{[9, 18-27]} Therefore, the microbiological safety of our novel HBAL must be clarified before clinical trials.

 

In our previous study, healthy dogs were used to evaluate the microbiological safety of a simple BAL,^[5] but ALF dogs had a lower immunizing power against virus infection. Eight canines with D-galactosamine-induced ALF were treated by novel HBAL, and PERV transmission was detected to clarify the microbiological safety of this novel HBAL in the present study. Because pseudotyping of murine endogenous retroviruses is the major factor for PERV transmission^{[28, 29]} and dogs possess huPAR-1 and muPAR genes encoding PERV receptors, dogs may be infected by PERV through the same mechanism as humans.^[30-32] In addition, the tests of PERV RNA, DNA, capsid protein and RT activity can be used for the screening of retrovirus. Among these tests, PCR and RT-PCR are the most common and sensitive method.^[4] We therefore evaluated several pairs of primers described in previous publications^{[7, 8, 33]} by PCR and RT-PCR using the nucleic acid extracted from canine cells. Finally, three pairs of primers were chosen due to their high specificity for PERV detection in canine cells (data not shown) with a high sensitivity of 0.25 in a PK15 cell per 1×10⁴ cells for protease,^[8] 0.3 in a PK15 cell per 1×10⁵ cells for polymerase,^[19] and 0.25 in a porcine cell per 1×10⁵ cells for SsCytB,^[8] respectively.

 

During the screening of blood samples, positive PERV RNA, DNA and RT activity were only detected in the 3rd circuit during the treatment. The presence of PERV RNA and RT activity demonstrated the PERV replication in the plasma of the 3rd circuit,^[34] while the negative detection for the samples from the 1st circuit, the 2nd circuit and the canine blood implied no virus transfer through the semipermeable membrane between plasma component separators and canine blood. Our results were consistent with the Kuddus' report that RT activity and the presence of PERV RNA are limited to the shell of the bioreactor in the bioartificial liver support system (BLSS).^[20] Although very low PERV DNA concentration was occasionally detected in the luminal effluent without PERV RNA on days 1 and 5, no PERV RNA transmission was observed. Similarly, a developed three-compartment BAL did not exhibit PERV permeation after 8-hour treatment.^[25] In addition, the absence of protease, polymerase and SsCytB genes in the DNA from canine PBMCs demonstrated no microchimerism in blood cells. In terms of tissue samples, no PERV DNA, RNA, RT activity and capsid protein gag p30 were detected in tissue lysates. These results further revealed no PERV transmission into animal viscera. Therefore, negative PERV detection in blood and tissues confirmed no infection of PERV in dogs.

 

In addition, in order to determine PERV infectivity in the system, we conducted in vitro infection experiments using HEK293 cells without the infection by collected plasma. Microchimeric cells were observed, similarly as reported that no infection is observed in HEK293 cells incubated with the supernatant of porcine hepatocytes and human fulminant liver failure plasma.^[35] In clinical trials, PERV infectivity in another two extracorporeal BAL systems such as the Academic Medical Center-BAL and HepatAssist system is detected by in vitro infection experiments, and no HEK293 cell is contaminated.^{[9, 18]} Moreover, we detected the production and infectivity of PERV from porcine hepatocytes cultured on the galactosylated chitosan nanofiber scaffolds, and neither enhanced viral expression nor HEK293 cell infection infected by the incubation of culture medium containing PERV was detected,^{[36, 37]} suggesting no obvious infectivity of PERV in our novel HBAL.

 

Nyberg et al^[38] compared three kinds of membrane with various pore sizes in typical BAL and showed that the pores at the diameter of 200 nm allow the infectious virus to across the membrane at the first day, while the pores with the cutoff of 400 kDa or 70 kDa (approximately 5 nm or 10 nm) allow the transfer of viral RNA at the 3rd and 7th day in spite of no infectious virus. Therefore, PERV transmission is highly correlated with the pore size, the compositions of semipermeable membrane, and the exposure duration of PERV. Except a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds, another component of our BAL system is the plasma component separator, the features of which are as follows: (1) smaller loss of high-molecular-weight substances; (2) plasma substitute fluid saving due to the small inside volume of 140 mL; (3) being with the semipermeable membrane of 10 nm pore size giving rise to high-volume selective plasma exchange; (4) being made from ethylene vinyl alcohol copolymer resin EVOH with excellent biocompatibility. Moreover, BAL treatment time was another important factor influencing on PERV transmission. Nyberg et al^[38] demonstrated that the extraluminal HEK293 cells were infected by the intraluminal media containing PERV after the exposure for 7 days in an in vitro study when the membrane porosity the two lumen was 70 kDa. In the previous trial,^{[9, 22]} the duration of BAL treatment was arranged from 4 to 35 hours, and there was no evidence for patient infection of PERV. In this study, exposure of the recipient plasma was 6 hours, which would lower the risk for PERV transmission. Thereby, it is reasonable to speculate that PERV-specific indicators were negative in blood and tissues of the dogs, and no PERV could transport across the plasma component separator during the treatment phase. Accordingly, no PERV infection attributed to no direct interaction among porcine hepatocytes and canine blood, pore size of semipermeable membrane in plasma component separator, short duration of BAL treatment and a low infectivity of PERV in the novel HBAL. However, longer treatment might be needed for patients with liver failure; thereby further research in clinic trial has been planned.

 

In conclusion, no obvious evidence regarding PERV infection in the dogs subjected to the application of our novel HBAL suggested that our novel HBAL system is safe within the therapeutic window of 6 hours. The microbiological safety provides a theoretical support of its clinical application in the future. Since the HBAL treatment is a more complicated process, the microbiological safety of the novel HBAL in clinical application needs to be investigated and evaluated further.

 

 

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