A Novel Bioartificial Liver System Based On Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) For Treatment Of Swine Acute Drug-induced Liver Failure

Objective:1.To optimize the isolation and purification method of human umbilical cord mesenchymal stem cells(hUCMSCs),and to identify their biological characteristics;2.To explore the method of expanding hUCMSCs using a hollow fiber bioreactor,and to evaluate cell expansion and derivation 3.Construct a new artificial liver system based on hUCMSCs and evaluate its operation;4.Evaluate the rescue effect of the new artificial liver system based on hUCMSCs on swine acute druginduced liver failure model.Methods:1 The primary cells of hUCMSCs were extracted by tissue block method,and then gradually expanded and purified.2 The proliferation ability of hUCMSCs was evaluated by CCK-8,the cell culture was observed,and hUCMSCs were induced to multiply differentiation,flow cytometry to identify cell surface markers.3 We used hollow fiber bioreactors to culture and expand hU-MSCs.4 We collected bioreactor medium to extract exosomes,then detected exosome particle size,surface markers,concentration,etc.5 The new artificial liver system of hUCMSCs was evaluated for its stability and safety by testing its operation.6 We evaluated the efficacy and safety of the novel artificial liver system in the treatment of acute drug-induced liver failure in pigs by observing the general condition,survival time,liver function,coagulation function,histology and other aspects of experimental animals.Results:1 The tissue block method can efficiently extract hUCMSCs.By passage under controlled conditions,hUCMSCs with higher purity can be obtained at P3.The CCK-8 proliferation test shows that hUCMSCs have good proliferation ability and can be used for subsequent research.2 The extracted hUCMSCs has high expression of CD73,CD90,CD 105 and low expression of CD45,CD11b,CD 19,CD34 and HLA-DR,and has the ability of adipogenic,osteogenic and chondrogenic triple line differentiation.3 hUCMSCs can be well attached to the surface of hollow fiber,and can be cultivated and proliferated by hollow fiber bioreactor.4.The medium containing high concentration of exosome was obtained by culturing hUCMSCs in hollow fiber bioreactor.The size of exosome was 50-130nm,and it had bilayer membrane structure.CD63,CD9,CD81,Syntenin and TSG101 were positive on exosome and hUCMSCs,while Calnexin was positive in hUCMSCs and negative in exosome.5 There was no significant difference in the total protein content between the outer lumen of the hollow fiber tube and the exudate of the inner lumen of the hollow fiber tube by BCA method.6 The relevant parameters of the new bioartificial liver system based on hUCMSCs are stable during operation,and the vital signs of the experimental animals are stable during the process of operation.7 The new artificial liver system can effectively reduce the transaminase,bilirubin levels of the pig liver failure model 8.The new bioartificial liver system can significantly inhibit the necrosis of hepatocytes and promote the proliferation and regeneration of hepatocytes;9.hUCMSCs exerts the above biological functions in a dose-dependent manner.Conclusion:In this study,through a series of experiments from isolation to inoculation and culture,it was proved that the hollow fiber bioreactor can be used as a good means of culture and expansion of hUCMSCs.On this basis,a novel bioartificial liver system based on hollow fiber bioreactors containing hUCMSCs can effectively control the development of acute drug-induced liver failure in pigs,promote liver regeneration,significantly prolong the survival time by regulating the inflammatory cascade and homeostasis pathways.