Exploration Of The Potential Of Dedifferentiation And Redifferentiation Of Primary Mouse Hepatocytes With The Technology Of Three-dimensional(3D) Bioprinting

Background:Liver has the ability of regeneration,which could be realized by cell dedifferentiation and redifferentiation after being damaged.This physiological process requires the precise regulation of various signaling pathways.Many signals play an important role in the pathway of liver regeneration,for example,YAP pathway is involved in the dedifferentiation of liver cells,and Wnt pathway works in the redifferentiation.Previous studies on the mechanism were limited to 2D planar cell culture model.In this study,we introduce a 3D bioprinting technique.This technique can help verify the conclusions obtained in 2D planar culture so as to gain more meaningful results,and explore the effect of 3D cell culture mode on hepatocyte regeneration.Method:In this study,primary mouse hepatocytes were isolated from mice,and 3D tissue of primary mouse hepatocytes were prepared.The 3D tissue was cultured in groups after the cell markers and cell activity were validated.The controlled group was cultivated with the improved standard medium,and the experimental group was cultivated with experimental medium that has 3mM more CHIR99021 medium than the controlled group.The tissue was cleaved and RNA was extracted at day 1,3,5,7,9,11,13 after bioprinting.Transcriptomic methods,including single-cell sequencing and real-time polymerase chain reaction(RT-qPCR),were used to elucidate the expression of liver function,dedifferentiation,and redifferentiation genes.Results:Primary mouse hepatocytes were successfully extracted from mouse liver.Using 3D bioprinting technology,the 3D tissue of primary mouse hepatocytes was successfully constructed.The cell viability was satisfied,and the staining results of hepatocyte markers was obvious.A total of 14 groups of RNA from 3D tissue of primary mouse hepatocytes and 2 groups of RNA from 2D culture of primary mouse hepatocytes were collected.Five genes,including Alb,Cyr61,Myc,p53 and Afp,were further verified by RT-qPCR.The expression level of Cyr61 gene in 2D culture was significantly higher than that in 3D culture(P=0.0137),but there was no statistical significance among other groups.Using single-cell sequencing,we collected three groups of cells,namely 3D tissue,2D cultured mouse hepatocytes and newly isolated mouse hepatocytes.A total of 10 genes were detected,including Alb,Cyp2d26,Hnf4a,Ctgf Sox9,Krtl9,Afp,Myc and Pml.We found that there was no obvious difference in the expression of genes related to liver function among the three groups of cells.Newly isolated mouse hepatocytes showed stronger dedifferentiation and activation of YAP pathway signals,while 2D cultured mouse hepatocytes showed more markers of liver progenitor cells,and 3D tissue moderated these activated pathways.Conclusion:This study is the first one to apply 3D bioprinting technology to study the cellular mechanism.Firstly,we established a stable and efficient primary mouse hepatocytes line,which has great implications for future cell research.Secondly,this study focuses on the dedifferentiation and redifferentiation potential of hepatocytes,which is of great significance to the understanding of the mechanism of liver regeneration and the realization of artificial liver in the future.According to transcriptomics,hepatocyte dedifferentiation begins when hepatocytes are isolated from animals and the YAP pathway is activated,then cells are converted into liver progenitor cells in continuous 2D culture.3D bio-printing technology changed the growth environment of cells,so that the dedifferentiated hepatocytes began to differentiate gradually.The whole tissue was able to maintain the expression of liver function genes during the experiment.At the same time,the regeneration process of liver still needs to be clarified by more studies,which may provide a new idea for drug design of liver dysplasia diseases.