Experimental Study On Immunological Safety Of A Novel Bioartificial Liver System

Objective: Given xenogeneic immune reaction is relevant to molecular weight cut offof the membrane of a bioartificial liver system (BAL), we investigated the influenceof membrane molecular weight cut off in our BAL system in this study.Methods: Acute liver failure in beagles was induced by D-galactosamineadministration. The new BAL was based on the new multi-layer radial-flowbioreactor containing galactosylated chitosan nanofiber scaffolds and co-system ofporcine hepatocytes and MSCs. Beagles were divided into two groups by themembrane molecular weight cut off of plasma component separator. GroupA beagleswere treated with BAL containing200kDa retention rating membrane. GroupBbeagles were treated with BAL containing1200kDa retention rating membrane. Eachgroup underwent two6-hour BAL treatments that were performed on day1and day21. The hemodynamic and hematologic response, humoral immune responses andcytotoxic immune response to BAL therapy were studied before and after treatments.Immunochemical staining of heart, liver, spleen, lung and kidney with IgG、IgM andC3antibodies were performed to determine deposition of canine proteins on thetissues.Results: Immediately after the first BAL treatment, groupA and groupB showedsimilar levels of IgG and IgM before and after treatment. However, we found thatbeagles experienced a significant increase in levels of IgG and IgM after two BALtreatments if membranes with1200kDa retention rating, but not200kDa retentionrating were used. In addition, we found that BAL treatment was associated with asignificant decline in levels of CH50; the time of level reaching to the lowest was60min. The level recovered to baseline on day7after treatments. Significant levels ofcanine proteins were detected in BAL medium from groupB, only trace levels ofcanine proteins were detected in BAL medium from groupA. No IgG or IgM orcomplement proteins were detected in the tissues from heart, liver, spleen, lung and kidney.Conclusion: Xenogeneic immune response was influenced by membrane molecularweight cut off in the BAL. Objective: Immunoisolation using semipermeable membranes has been incorporatedinto BAL to separate celluar components of the recipient’s immune system from thecells within the BAL device. This study was designed to explore the influence ofmembrane molecular weight cut off on performance of the multi-layer radial-flowBAL using porcine hepatocytes co-cultured with MSCs.Methods: The new BAL was based on the new multi-layer radial-flow bioreactorcontaininggalactosylated chitosan nanofiber scaffolds and co-system of porcinehepatocytes and MSCs. To investigate whether freshly isolated porcine hepatocyteand MSCs express Galα(1,3) Gal, immunofluorescene and RT-PCR were performedon freshly isolated porcine hepatocyte and MSCs. Healthy beagles underwent6-hourtreatment with a BAL containing membrane with200kDa retention rating or1200kDa retention rating. Functional markers of BAL performance were monitoredbefore and after treatment, as well as cytotoxic immune response to BAL therapy.Results: Freshly isolated porcine hepatocyte and MSCs express Galα(1,3) Gal.Hepatocyte performance levels such as albumin secretion and urea synthesis werewere all significantly higher in200kDa retention rating group compared with1200kDa retention rating group after treatment (p<0.05). Viability of co-culture cellsin1200kDa group decreased from91%to22%. In contrast, the viability ofco-culture cells was stable at90%in200kDa group. The AST release in1200kDagroup slightly increased from13.3U/L to60.2U/L, however, the level in200kDagroup was stable at13.2U/L to14.8U/L. The level of IgG was detected in BALmedium obtained30min after initiation of treatment in1200kDa group, and then itincreased from0.4at30min to2.7at6hr. There was a significant difference between the two groups over6hr (P＜0.05). Similar patterns were observed for CH50levels.The level of IgM in BAL medium was undetectable during the course of treatment ingroup1, in contrast, the BAL medium from1200kDa group contained detectable levelof IgM in30min after treatment, and it increased from0.7±0.02at30min to2.3±0.08at6hr. There was a significant difference between the two groups over6hr (P＜0.05).Fluorescence microscopy further verified that heavy deposition of canine IgG,IgM and complement (C3) on co-culture cells obtained after BAL treatment in1200kDa retention rating group. However, only trace deposits of canineimmunoproteins were observed on co-culture cells obtained from BAL in200kDaretention rating group.Conclusion: Small membrane molecular weight cut off of the BAL could reduce thetransfer of xenoreactive antibodies into the BAL medium and improve performanceof the BAL Objective: To study and evaluate the immunosafety of our newly developedmulti-layer flat-plate bioartificial liver in treatment of canines with acute liver failure.Methods: Fresh porcine hepatocytes and bone marrow mesenchymal stem cells werecocultured in our new bioartificial liver, and the separator contains a membrane with200kDa retention rating.10canine models with acute liver failure were set upthrough D-galactosamine administration.24hours after administration, the beagleswere randomly allocated to a6-hour treatment with the bioartificial liver. The beagleswere divided into two groups by treatment times. Group1beagles (n=5) received asingle BAL treatment. Group2beagles (n=5) received three BAL treatments. Thehemodynamic, hematologic response and humoral immune responses to BAL therapywere studied before and after treatments. Levels of IgG, IgM and CH50were detectedby ELISA kit. Tissues from heart, liver, spleen, lung and kidney were examined byepifluorescence microscopy for evidence of immune complex deposition using fluorescence-labeled antibodies.Results: All beagles remained hemodynamically and hematologically stable duringBAL treatment. The levels of IgG and IgM were similar before and after treatmentafter a single treatment. In addition, the level of CH50in group1slightly decreasedafter the initiation of BAL treatment, and then the level recovered to baseline quicklyafter treatments. Time-course changes of the levels of antibodies and CH50after threetreatments in group2were similar to group1. Only trace levels of IgG and CH50weredetected in BAL medium after treatments. The level of IgM in BAL medium wasundetectable during the course of treatment. Only trace deposits of IgG and C3wereobserved on co-culture cells obtained from BAL. No IgG or IgM or C3were detectedin the tissues from heart, liver, spleen, lung and kidney. No evidence for an immunecomplex glomerulonephritis was observed.Conclusion: The multi-layer flat-plate bioartificial liver showed a greatimmunosafety in the treatment of canines with acute liver failure and exhibited agood prospect of its use in clinic.