Construction Of Three-dimensional Bioprinted Liver Organoid And Its Protein Synthesis Function

Background:The repair and maintenance of liver function is key to the treatment of patients with advanced liver disease and liver cancer,while the protein synthesis is one of the most crucial part of liver function,among which albumin and coagulation factors are quite essential in clinical applications.At present,the main sources of albumin and coagulation factors under clinical situations are the isolation and purification from peripheral blood products or the expression of recombinant proteins in mammalian cell system.Still,the demand exceeds supply.The purpose of this research is to apply the widely used extruding 3D bioprinting technology to bioenginnering field.The mix of gelatin,sodium alginate and HepaRG cell suspension serves as the bio-ink.After bioprinting and shaping,a 3D bioprinted liver organoid is built,and its protein synthetic function is studied to further explore the potential clinical application value of using bioprinted organoid for protein synthesis invitro.Method:We used HepaRG cell,which is the most widely used immortalized human stem-cell like hepatocyte,as the main cell component.The gelatin and sodium alginate were used as scaffolds of the bio-ink.The liver organoid model was established by bioprinting using the mixed bio-ink,as well as optimizing printing parameters and cell culture conditions.We compared 2D cultured HepaRG cells with 3D bioprinted HepaRG organoid.During the culturing period,we recorded the morphologic changes of each cell group over time,collected the supernatant and printed tissues,and performed transcriptomic sequencing analysis,quantitative measurements of albumin and coagulation factors,coagulation factor activity assays and proteomic analysis.Result:This experiment compared the transcriptomic sequencing results of any two groups among the 2D cells before differentiation induction,2D cells after differentiation induction and 3D bioprinted organoid.Significantly different gene expression profiles were revealed in cell components,biological processes and molecular functions.The cluster analysis and protein-protein interaction analysis of the differentially expressed genes were also performed.At the same time,we measured the total protein in the supernatant of the 2D culture,3D culture,and 3D printed group,and determined the content of albumin and coagulation factors.We found that most results in 3D printed group were higher than 2D culture or 3D culture.We also preliminarily tested the coagulation factor activity of the 3D bioprinted organoid.In addition,starvation tests were conducted in each group to lay the foundation for the proteomic analysis at the next step.Conclusion:In this research,a 3D bioprinted liver organoid model was constructed,and the protein synthesis function of this model was explored from the level of transcriptomic analysis,protein quantitative analysis and proteomic analysis.The construction of organoids by 3D bioprinting technology,understanding the synthesis and secretion functions of liver organoids and exploring other proteins highly expressed in liver organoids through transcriptomic and proteomic analysis can help us further discover its potential applications and clinical translational value.