Jinyang Gu

In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds

Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.

Coencapsulation of hepatocytes with bone marrow mesenchymal stem cells improves hepatocyte-specific functions.

Background. Hepatocyte transplantation is an alternative to liver transplantation, which is hampered by short survival time and immunorejection. In this study, we investigated the specific functions of hepatocytes coencapsulated with bone marrow mesenchymal stem cells (MSCs) in vitro, and we further examined whether transplantation of coencapsulated hepatocytes and MSCs could enhance the ability of hepatocytes to alleviate acute liver failure in vivo. Methods. Rat hepatocytes and MSCs were isolated and coencapsulated by alginate-poly-l-lysine-alginate microencapsulation. Cell functions were monitored during the course of cell culturing. Acute liver failure in rats was induced by d-galactosamine administration. These rats were subjected to intraperitoneal transplantation of coencapsulated hepatocytes with MSCs, encapsulated hepatocytes alone, or empty vehicles after 24 hr. The survival rate and liver functions were assessed. Immunofluorescence microscopy was used for the analysis of coencapsulated cells before and 7 days after transplantation. Results. Hepatocyte-specific functions, including albumin secretion and urea synthesis, were all significantly improved in the coencapsulation group compared with the encapsulated hepatocytes group (P<0.05). Similar trend was observed for cell cycle analysis of hepatocytes. Intraperitoneal transplantation of coencapsulated hepatocytes with MSCs not only increased the survival rate but also improved liver functions in a rat model of acute liver failure. Some MSCs transdifferentiated into hepatocyte-like cells in vivo, which expressed albumin, a typical marker of hepatocyte. Conclusions. Encapsulation of hepatocytes and MSCs improved hepatocyte-specific functions in vitro and in vivo. Transplantation of coencapsulated hepatocytes and MSC might be a promising strategy for cell-based therapy for acute liver diseases.

Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro.

Temporary replacement of specific liver functions with extracorporeal bioartificial liver has been hampered by rapid de‐differentiation of porcine hepatocytes in vitro. Co‐cultivation of hepatocytes with non‐parenchymal cells may be beneficial for optimizing cell functions via mimicry of physiological microenvironment consisting of endogenous matrix proteins. However, the underlying mechanisms remain to be elucidated. A randomly distributed co‐culture system composed of porcine hepatocytes and bone marrow mesenchymal stem cells was generated, and the morphological and functional changes of varying degrees of heterotypic interactions were characterized. Furthermore, contributions of extracellular matrix within this co‐culture were evaluated. A rapid attachment and self‐organization of three‐dimensional hepatocyte spheroids were encouraged. Studies on hepatocyte viability showed a metabolically active, viable cell population in all co‐culture configurations with occurrence of few dead cells. The maximal induction of albumin production, urea synthesis, and cytochrome P4503A1 activities was achieved at seeding ratio of 2:1. Immunocytochemical detection of various extracellular matrix confirmed that a high level of matrix proteins synthesis within distinct cells was involved in hepatocyte homeostasis. These results demonstrate for the first time that cell–matrix has synergic effects on the preservation of hepatic morphology and functionality in the co‐culture of porcine hepatocytes with mesenchymal stem cells in vitro, which could represent a promising tool for tissue engineering, cell biology, and bioartificial liver devices. J. Cell. Physiol. 219: 100–108, 2009.

Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro

Aim:  The application of porcine hepatocytes in liver support systems has been hampered by the short‐term survival. Co‐cultivation of hepatocytes with non‐parenchymal cells may be beneficial for optimizing cell functions via heterotypic interactions. In this study, we present a new cultivation system of porcine hepatocytes and mesenchymal stem cells (MSCs) in a randomly distributed co‐culture manner.

In vitro analysis of cryopreserved alginate–poly-l-lysine–alginate-microencapsulated human hepatocytes

Background: The availability of well‐characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications.

Efficacy and safety of liver transplantation in patients with cholangiocarcinoma: A systematic review and meta‐analysis

The aim of our study was to evaluate the efficacy and safety of liver transplantation in patients with cholangiocarcinoma. According to the requirements of Cochrane systematic review, a thorough literature search was performed in PubMed/Medline, Embase and Cochrane electronic databases between 1995 and 2009 in terms of the key words “liver transplantation” and “cholangiocarcinoma,” “cholangiocellular carcinoma” or “bile duct cancer,” with restricted articles for the English language. Data were processed for a meta‐analysis by Stata 10 software. Altogether 14 clinical trials containing 605 transplanted patients of bile duct cancers were finally enrolled in our study. The overall 1‐, 3‐ and 5‐year pooled survival rates were 0.73 [95% confidence interval (CI) = 0.65–0.80], 0.42 (95% CI = 0.33–0.51) and 0.39 (95% CI = 0.28–0.51), respectively. Of note, preoperative adjuvant therapies [orthotopic liver transplantation (OLT)‐PAT group] rendered the transplanted individuals with comparably favorable outcomes with 1‐, 3‐ and 5‐year pooled survival rates of 0.83 (95% CI = 0.57–0.98), 0.57 (95% CI = 0.18–0.92) and 0.65 (95% CI = 0.40–0.87). In addition, the overall pooled incidence of complications was 0.62 (95% CI = 0.44–0.78), among which that of OLT‐PAT group (0.58; 95% CI = 0.20–0.92) was relatively acceptable compared to those of liver transplantation alone (0.61; 95% CI = 0.33–0.85) and liver transplantation with extended bile duct resection (0.78; 95% CI = 0.55–0.94). In comparison to curative resection of cholangiocarcinoma with the 5‐year survival rate reported from 20 to 40%, the role of liver transplantation alone is so limited. In the future, attention will be focused on liver transplantation following neoadjuvant radiochemotherapy, which requires a well‐designed, prospective randomized controlled study.

Evaluation of a novel hybrid bioartificial liver based on a multi-layer flat-plate bioreactor.

AIM To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure. METHODS Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected. RESULTS Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 μmol/L vs 24.2 ± 6.45 μmol/L, 12.47 ± 3.62 μmol/L, 3.77 ± 1.83 μmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative. CONCLUSION The HBAL showed great efficiency and safety in the treatment of acute liver failure.

Microbiological safety of a novel bio-artificial liver support system based on porcine hepatocytes: a experimental study

BackgroundOur institute has developed a novel bio-artificial liver (BAL) support system, based on a multi-layer radial-flow bioreactor carrying porcine hepatocytes and mesenchymal stem cells. It has been shown that porcine hepatocytes are capable of carrying infectious porcine endogenous retroviruses (PERVs) into human cells, thus the microbiological safety of any such system must be confirmed before clinical trials can be performed. In this study, we focused on assessing the status of PERV infection in beagles treated with the novel BAL.MethodsFive normal beagles were treated with the novel BAL for 6 hours. The study was conducted for 6 months, during which plasma was collected from the BAL and whole blood from the beagles at regular intervals. DNA and RNA in both the collected peripheral blood mononuclear cells (PBMCs) and plasma samples were extracted for conventional PCR and reverse transcriptase (RT)-PCR with PERV-specific primers and the porcine-specific primer Sus scrofa cytochrome B. Meanwhile, the RT activity and the in vitro infectivity of the plasma were measured.ResultsPositive PERV RNA and RT activity were detected only in the plasma samples taken from the third circuit of the BAL system. All other samples including PBMCs and other plasma samples were negative for PERV RNA, PERV DNA, and RT activity. In the in vitro infection experiment, no infection was found in HEK293 cells treated with plasma.ConclusionsNo infective PERV was detected in the experimental animals, thus the novel BAL had a reliable microbiological safety profile.

Effects of Membrane Molecular Weight Cutoff on Performance of a Novel Bioartificial Liver

Immunoisolation using semipermeable membranes has been incorporated into bioartificial liver (BAL) devices to separate cellular components of the recipients immune system from the cells within the BAL device. This study was designed to explore the influence of membrane molecular weight cutoff on performance of the multilayer radial-flow BAL using porcine hepatocytes cocultured with mesenchymal stem cells. In this study, healthy beagles underwent 6-h treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating. Functional markers of BAL performance were monitored before and after treatment, as well as cytotoxic immune response to BAL therapy. The results showed that hepatocyte performance levels such as albumin secretion, urea synthesis, and viability were all significantly higher in 200 kDa retention rating group compared with the 1200 kDa retention rating group after treatment (P <  0.05). Significant levels of canine proteins were detected in BAL medium from the 1200 kDa retention rating group. Fluorescence microscopy further verified that heavy deposition of canine IgG, IgM, and complement (C3) on coculture cells was obtained after BAL treatment in the 1200 kDa retention rating group. However, only trace deposits of canine immunoproteins were observed on coculture cells obtained from BAL in the 200 kDa retention rating group. Small membrane molecular weight cutoff of the BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improve the performance of the BAL.

The Influence of Membrane Molecular Weight Cutoff on a Novel Bioartificial Liver

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.