The Generation Of An Engineered Liver Based On Liver Bud Formation

Background & Aims:Liver transplantation remains the most effective treatment for end-stage liver disease.However,due to the shortage of donor liver,a large number of patients with liver failure can not be treated promptly.With the development of regenerative medicine,liver tissue engineering has brought fresh hope to millions of sufferers.Liver tissue engineering,also known as engineered liver,based on the concept of tissue engineering is aiming to grow replacement organs for transplantation into patients and create tissues that can be used in the lab to model human disease and test potential new drugs.While scientists are still targeting that goal,a series of technical problems,such as the source of seed cells,the construction of three-dimensional structure,the reconstruction of vascular network and bile duct structure,have not yet been solved thoroughly,which restricts the development of liver tissue engineering.To date,no one has yet come to clinical practice.In this study,a new type of seed cell source for liver tissue engineering was discovered through the proliferation of human primary hepatocytes in vitro.What’s more,based on the formation of liver buds,we provided a new idea for the construction of liver tissue engineering with physiological functions and three-dimensional structure.Methods:1.Primary human hepatocytes were isolated by two-step perfusion method.The Transition and Expansion Medium(TEM)was optimised for cell expansion,and then the proliferative hepatocytes were identified by flow cytometry and so on;2.HepLPCs,HUVECs and hUC-MSCs were mixed and cultured in vitro to formed liver buds,and their culture conditions were explored and optimized through observation;3.The cell viability,hepatocyte functions and the bipotential differentiation of the liver bud were determined by means of ELISA,CCK8,PCR,biochemical test and so on;4.Decellularized liver matrix were prepared by low temperature chemical methods and examined for their biological composition,morphology and cytotoxicity;5.The above three kinds of cells were mixed and put into the decellularized liver scaffold to construct an engineered liver,and then the liver was cultured by circulatory perfusion;6.The engineered liver constructed in vitro were evaluated for liver function and histology at different time points.Results:Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells through delivery of developmentally relevant cues.We found that the HepLPCs can be mixed with HUVECs and hUC-MSCs to form liver buds in vitro.Based on the formation of liver bud,we put the three kinds of cells into the decellularized liver scaffold to construct an engineered liver with three-dimensional structure and physiological function through continuous perfusion culture.Conclusions:Our work demonstrates the utility of the conversion between human primary hepatocyte and liver progenitor-like cells for constructing liver tissue engineering,which is of great scientific significance for regenerative medicine.