The Establishment And Validation Of Liver Related In Vitro Model Based On 3D Bioprinting

Background:With the rapid development of 3D bioprinting,it has become one of the most critical technology for fabricating in vitro tissues and organs.3D bioprinted human tissues and organs,including skin,cartilage,pancreas,kidney,lung and heart,have subsequently emerged with nearly the same morphological characteristics and biological functions as that in vivo.As the most crucial digestive gland,the liver plays a vital role in biosynthesis,drug metabolism and blood coagulation.Many liver-related diseases have become the main threats to human health,such as drug-induced liver injury,viral hepatitis,cirrhosis and liver cancer.However,there are no eligible in-vitro research models for liver-related diseases.Therefore,this study aims to construct 3D bioprinted models for liver and liver cancer,and to further verify its practical value.Methods:To construct a 3D bioprinted liver model,we used HepaRG as the seeding cell while gelatin/sodium alginate as the bio-ink to fabricate a latticed in-vitro human liver tissue(3D bioprinted hepatorganoids,3DP-HOs)by an extrusional 3D bioprinter.Firstly,we tested the best parameters for 3D bioprinting and subsequent cell culture of bio-ink composition,temperature and procedures of cell induction procedures.Then,to verify the success of the model,we examined morphological characteristics and viability(cell survival staining and CCK8 proliferation test)of 3DP-HOs.After creating the liver model,we designed five groups as primary human hepatocytes,undifferentiated HepaRG,2D cultured HepaRG,3D cultured HepaRG and 3DP-HOs.We compared level of liver-specific genes and proteins among the groups using qPCR,ELISA and immunostaining.PAS staining,ICG uptake&excretion test and LDL uptake test were carried out to evaluate the specific functions of 3DP-HOs.Besides,all the groups were separately induced by 3-methylcholesterol,phenobarbital sodium and rifampicin to measure the gene expression and activity of multiple CYP450 enzymes using qPCR and P450-GloTM Assays before and after induction.Finally,we transplanted 3DP-HOs into the abdomen of Fah-/-Rag2-/-mice with liver injury.We evaluated the in-vivo function of the 3D bioprinted liver by analyzing the integration of 3DP-HOs in the body,debrisoquine metabolism,physiological indicators of liver function and murine survival.To simulate liver cancer,HepG2 and gelatin-sodium alginate were used to construct a latticed model(3D bioprinted HepG2,3DP-HepG2)using the same extrusional 3D bioprinter as before.Morphological characteristics and cell survival status were employed as the indicators of effectiveness of the model,in which Ki67 was used as a marker to evaluate the cell proliferation.Between the two groups of 3DP-HepG2 and 2D-cultured cells,we used qPCR and immunostaining to detect level of genes and proteins specific to liver,liver cancer,autophagy,and drug-resistance.Transcriptome sequencing and bioinformatics analysis were conducted to analyze overall gene profiles and screen core differential genes between groups.Finally,we evaluated different drug responses between the 3DP-HepG2 and 2D cultured group through the drug test of cisplatin,sorafenib and regorafenib.Results:4%gelatin/1%sodium alginate was finally selected as the bio-ink,and latticed liver tissue(10×10×3mm3)was successfully constructed under 10?(temperature of printing room)/20?(temperature of material room).During long-term culture,3DP-HOs maintained stable proliferation and more than 90%of the cell viability.After 7 days of induction,3DP-HOs contained 82.5%±4.6%of ALB-positive hepatocytes,and expressed higher levels of ALB,AAT,CK18,MRP2,Transferrin,FOXA2 and HNF4A than other groups of undifferentiated HepaRG,2D&3D cultured HepaRG.Results of secreted protein expression and immunofluorescence coordinated with each other.CYP induction tests showed that gene expression,including CYP1A2,CYP2A6,CYP2B6,CYP2C8,CYP2C9,CYP2D6,CYP3A4 and CYP3A11,were enhanced in 3DP-HOs under specific drug induction.Especially the activities of CYP1A2 and CYP3A4 were increased by more than two-fold after induction.In addition,3DP-HOs displayed the capacity of glycogen storage,ICG uptake&excretion and LDL uptake.After transplantation of 3DP-HOs into mice,3DP-HOs ameliorated liver injury in Fah--Rag2-/-mice,lowered the level of various amino acids,and ultimately prolonged the survival of mice.It was found that a vascular network was formed in the transplanted 3DP-HOs,which could secrete human ALB,AAT,Coagulation factor ?,? and ? after 4 weeks of transplantation.And 3DP-HOs could metabolize debrisoquine in vivo.3DP-HepG2 also has a size of 10×10×3mm3 and the cell in the model maintained a high cell viability of about 90%and strongly positive Ki-67 expression from the first day to the 10th day after printing.Compared with 2D-HepG2,3DP-HepG2 expressed a higher level of genes,including those specific to liver like ALB,AAT,CYP3A4,and those specific to tumor-like AFP,CD133,EpCAM,IL8 and CD24.Transcriptome sequencing showed that 3DP-HepG2 had a unique gene expression profile,with core differential genes of ALB and NOTUM,and significant difference in genes of the liver function and tumor from 2DP-HepG2.Drug tests showed that the IC50 of cisplatin,sorafenib and regorafenib in 3DP-HepG2/2D-HepG2 were respectively 38.56?M/12.03?M,22.07?M/6.53?M and 7.93?M/1.96?M.The qPCR results showed that 3DP-HepG2 had higher expression of genes related to drug resistance(MRP 1,ACBC1,MDR1,EGFR)and autophagy(Beclin3-1,LC3A,LC3B and Atg5).Conclusions:3D bioprinted liver tissue could be employed as a competent model in liver protein synthesis and drug metabolism,which could ameliorate liver injury and prolong survival in mice with hepatic failure,showing a promising future of transplantation treatment.3D bioprinted liver cancer model has biological characteristics specific to liver and tumor,which could be potentially used in drug screening.