Construction Of A Novel In Vitro 3D Hepatocyte Model Based On Reversible Conversion And 3D Culture Of Hepatocytes

OBJECTIVESHepatocytes serve as important tools in drug toxicity screening and disease modeling in vitro,but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and functions.Therefore,based on our previous research,we aim to establish an immortalized liver progenitor-like cell(i Hep LPC)and its associated differentiation protocol to provide a reliable and practical hepatocyte model for the hepatocyte industry.METHODSWe analyzed the proliferation characteristics of iHepLPCs by proliferation curve,doubling time and karyotype analysis,and analyzed the differentiation ability of modified hepatocyte maturation medium(m HMM)for 2D and 3D cultured iHepLPCs using QPCR,PAS and biochemical assays.Further,we characterization these cells by RNA-Sequence.We then used our 3D hepatocyte model to examine the hepatotoxicity of a range of hepatotoxic drugs and compare their toxic reaction with primary hepatocytes,Hep G2 and Hepa RG.To detect heterogeneity,we used 6different donor-derived iHepLPCs to detect the different toxic reaction of the same drug.Through these methods,we demonstrate the practicality and reliability of this model.In addition,QPCR,PAS,immunofluorescence,and biochemical assays were used to analyze the differentiation ability of Hepa RG induced by m HMM.The results indicating that m HMM also has broad application prospects.RESULTSiHepLPCs could efficiently expand for at least 40 population doublings,with a steady proliferative property.They were able to readily differentiate back into metabolically functional hepatocytes in vitro within 10 days.Furthermore,under three-dimensional culture condition,the formed hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes.Importantly,by generating a certain number of such cell lines,we established a hepatocyte bank with,to some extent,a population heterogeneity which allows us to analyze the in vitro heterogeneous responses in drug toxicity.Additionally,we found that m HMM can also induce the maturation of Hepa RG cells in a short time.These findings will facilitate the in vitro studies of human liver diseases and the industrial applications of human hepatocytes.CONCLUSIONSiHepLPCs-Hep-3D can be used as a reliable hepatocyte model for the development of the pharmaceutical industry and the bioartificial liver devices.m HMM,which is an important part of the differentiation system of iHepLPCs-Hep-3D,can also be used as an effective differentiation method for Hepa RG cell lines.