Hepatic reconstruction from fetal porcine liver cells using a radial flow bioreactor

AIM： To examine the efficacy of the radial flow bioreactor （RFB） as an extracorporeal bioartificial liver （BAL） and the reconstruction of liver organoids using embryonic pig liver cells. METHODS： We reconstructed the liver organoids using embryonic porcine liver cells in the RFB. We also determined the gestational time window for the optimum growth of embryonic porcine liver cells. Five weeks of gestation was designated as embryonic day （E） 35 and 8 wk of gestation was designated as E56. These cells were cultured for one week before morphological and functional examinations. Moreover, the efficacy of pulsed adminisbation of a high concentration hepatocyte growth factor （HGF） was examined. RESULTS： Both cell growth and function were excellent after harvesting on E35. The pulsed administration of a high concentration of HGF promoted the differentiation and maturation of these fetal hepatic cells. Microscopic examination of organoids in the RFB revealed palisading and showed that bile duct-like structures were well developed, indicating that the organoids were mini livers. Transmission electron microscopy revealed microvilli on the luminal surfaces of bile duct-like structures and junctional complexes, which form the basis of the cytoskeleton of epithelial tissues. Furthermore, strong expression of connexin （Cx） 32, which is the main protein of hepatocyte gap junctions, was observed. With respect to liver function, ammonia detoxification and urea synthesis were shown to be performed effectively. CONCLUSION： Our system can potentially be applied in the fields of BAL and transplantation medicine.

Ultrasonic characterization of porcine liver tissue at frequency between 25 to 55 MHz

AIM： To study the relation between acoustic parameters and histological structure of biological tissue and to provide the basis for high-resolution image of biological tissues and quantitative ultrasonic diagnosis of liver disease. METHODS： Ultrasonic imaging and tissue characterization of four normal porcine liver and five cirrhotic liver tissue samples were performed using a high frequency imaging system. RESULTS： The acoustic parameters of cirrhotic liver tissue were larger than those of normal liver tissue. The sound velocity was 1577 m/s in normal liver tissue and 1631 m/s in cirrhotic liver tissue. At 35 MHz, the attenuation coefficient was 3.0 dB/mm in normal liver tissue and 4.1 dB/mm in cirrhotic liver tissue. The backscatter coefficient was 0.00431 dB/Srmm in cirrhotic liver tissue and 0.00303 dB/Srmm in normal liver tissue. The backscatter coefficient increased with the frequency. The high frequency images coincided with their histological features. CONCLUSION： The acoustic parameters, especially the sound backscatter coefficient, are sensitive to the changes of liver tissues and can be used to differentiate between the normal and pathological liver tissues. High frequency image system is a useful device for highresolution image and tissue characterization.

Time-restricted feeding downregulates cholesterol biosynthesis program via RORγ-mediated chromatin modification in porcine liver organoids

Background: Time-restricted feeding(TRF) is a dieting strategy based on nutrients availability and diurnal rhythm,shown to improve lipid metabolism efficiency. We have demonstrated previously that retinoic acid-related(RAR)orphan receptor(ROR) γ is the primary transcription factor controlling cholesterol(CHO) biosynthesis program of animals. However, the functional role of RORγ in liver physiology of pigs in response to TRF has not been determined, largely due to the lack of functional models and molecular tools. In the present study, we established porcine liver organoids and subjected them to restricted nutrients supply for 10-h during the light portion of the day.Results: Our results showed that TRF regimen did not alter hepatocyte physiology, including unchanged cell viability, caspase 3/7 enzyme activity and the gene signature of cell proliferation in porcine liver organoids,compared to the control group(P > 0.05). Furthermore, we found that TRF downregulated the hepatic CHO biosynthesis program at both mRNA and protein levels, along with the reduced cellular CHO content in porcine liver organoids(P < 0.05). Using unbiased bioinformatic analysis of a previous ChIP-seq data and ChIP-qPCR validation, we revealed RORγ as the predominant transcription factor that responded to TRF, amongst the 12 targeted nuclear receptors(NRs)(P < 0.05). This was likely through RORγ direct binding to the MVK gene(encoding mevalonate kinase). Finally, we showed that RORγ agonists and overexpression enhanced the enrichment of cofactor p300, histone marks H3 K27 ac and H3K4me1/2, as well as RNA polymerase II(Pol-II) at the locus of MVK, in TRF-porcine liver organoids, compared to TRF-vector control(P < 0.05).Conclusions: Our findings demonstrate that TRF triggers the RORγ-mediated chromatin remodeling at the locus of CHO biosynthesis genes in porcine liver organoids and further improves lipid metabolism.

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.

Effect of atrial natriuretic peptide on ischemia-reperfusion injury in a porcine total hepatic vascular exclusion model

AIM： To evaluate the effect of ANP on warm I/R injury in a porcine THVE model.METHODS： Miniature pigs （mini-pigs） weighing 16-24 kg were observed for 120 min after reperfusion following 120 min of THVE. The animals were divided into two groups. ANP （0.1 μg/kg per min） was administered to the ANP group （n = 7）, and vehicle was administered to the control group （n = 7）. Either vehicle or ANP was intravenously administered from 30 min before the THVE to the end of the experiment. Arterial blood was collected to measure AST, LDH, and TNF-α. Hepatic tissue blood flow （HTBF） was also measured. Liver specimens were harvested for p38 MAPK analysis and histological study. Those results were compared between the two groups.RESULTS： The AST and LDH levels were lower in the ANP group than in the control group; the AST levels were significantly different between the two groups （60 min： 568.7 ± 113.3 vs 321.6 ± 60.1, P = 0.038 〈 0.05, 120 rain： 673.6± 148.2 vs 281.1±44.8, P = 0.004 〈 0.01）. No significant difference was observed in the TNF-α levels between the two groups. HTBF was higher in the ANP group, but the difference was not significant. A significantly higher level of phosphorylated p38 MAPK was observed in the ANP group compared to the control group （0min： 2.92± 1.1 vs 6.38 ±1.1,,P= 0.011 〈 0.05）.Histological tissue damage was milder in the ANP group than in the control group.CONCLUSION： Our results show that ANP has a protective role in I/R injury with p38 MAPK activation in a porcine THVE model.