Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

AIM： To observe the effects of plasma from patients with severe viral hepatitis （SVHP） on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS： Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione （GSH）, catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS： The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum （NCS） was significant in t-test （LDH： t = 24.552, P = 0.001; AST： t = 4.169, P = 0.014）. After exposure to SVHP for 24 h, alterations in GSH status were significant （F = 2.746, P〈0.05） between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h （F = 4.378, ,P〈0.05）. A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHR The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION： Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes, It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

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.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assaykits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.

Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system

BACKGROUND: The demand for the clinical use of hepa- tocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes. METHODS: The domestic extracorporeal circulating perfu- sion apparatus was used to isolate and harvest swine hepato- cytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and ob- served by the trypan blue exclusion test, biochemical mea- surements, phase contrast microscopy and transmission electron micrography (TEM). RESULTS: The total yield of isolated hepatocytes reached to 1.5(±0.4)×l010 per liver with a viability of 92(±5)%, and the purity of hepatocytes reached to 98% Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotrans- ferase ( AST ), lactate dehydrogenase ( LDH ), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly. CONCLUSION: This method of procuring swine hepato- cytes could get high quality cells with active metabolic function.

Functional evaluation of a new bioartificial liver system in vivo and in vivo

AIM:To evaluate the functions of a new bioartificial liver(BAL)system in vitro and in vitro.MEHTODS:The BAL system was configurated byinoculating porcine hepatocyte spheroids into the cellcircuit of a hollow fiber bioreactor.In the experiments ofBAL in vitro,the levels of alanine aminotransferase(ALT),total bilirubin(TB),and albumin(ALB)in the circulatinghepatocyte suspension and RPMI-1640 medium weredetermined during 6 h of circulation in the BAL device.In the experiments of BAL in vitro,acute liver failure(ALF)model in canine was induced by an end-side portocavalshunt combined with common bile duct ligation andtransaction.Blood ALT,TB and ammonia levels ofALF in canines were determined before and after BALtreatment.RESULTS:During 6 h of circulation in vitro,therewas no significant change of ALT,whereas the TB andALB levels gradually increased with time both in thehepatocyte suspension and in RPMI-1640 medium.Inthe BAL treatment group,blood ALT,TB and ammonialevels of ALF in canines decreased significantly.CONCLUSION:The new BAL system has the ability toperform liver functions and can be used to treat ALF.