A Research On The Three-dimentional Culture Technique Of Liver Tissue Mimicking The Hepatic Plate Structure

Background:Liver failure is a severe clinical syndrome, associated with high mortality of 60%. Liver transplantation is the only therapeutic way to liver failure, but it’s clinical application is seriously restricted due to the lack of donor, immune rejection. Bioartificial liver brings hope to the patients with liver failure. It can not only bridge patients over the transplantation, but also recover the liver function in patients with reversible liver failure to avoid liver transplantation.Nowadays, bioartificial liver is faced with the main problem that how hepatocytes can maintain hepatocyte phenotype and differentiated functions when cultured in vitro for long time by taking advantage of bioreactors. An important principle in improving hepatocytes’ function in vitro is to simulate the conditions of hepatocytes in the body. Therefore, many researchers propose the idea that three-dimensional culture is an effective way to solve the above problems.Numerous three-dimensional culture approaches have been developed to preserve hepatocyte functions in vitro. For example, hollow fiber-based culture, microcarrier culture, spheroidal aggregate culture, cell sheets, rapid prototyping-based culture, etc. However, all of the applications are faced with the same problem that hepatocytes are just cultured in three-dimensional condition and it’s not clear about the mechanism of constructing hepatocyte polarity including the co-culture of hepatocytes and non-parenchymal cells, the micro-tissues’ bionic arrangement, cell-cell and cell-matrix interactions, etc. Besides, the vitality and function are far from that in vivo.Actually, liver itself is a perfect bioreactor of hepatocytes in which hepatocytes meet the requirements of the quantity and density and are arranged into the hepatic plate structure and attain polarized. Recent studies have shown that the unique functioning of the liver is ensured by hepatocyte polarity.Based on the bionics and close relationship between polarity and function, three-dimensional culture of liver tissue mimicking the hepatic plate structure may become an effective way to solve the above problems. Becides, only by simulating the state in vivo to the greatest extent and simulating "hepatic lobule" and "hepatic plate" as well as combining this idea with bioreactors to realize its bionic design can we maintain hepatocytes’ full phenotype and differentiated functions in vitro.Objective:Based on the bionics that hepatocytes are arranged into the polarized hepatic plate structure and attain cell-cell and cell-matrix interactions within a liver, we propose the project of three-dimensional culture of liver tissue mimicking the hepatic plate structure. We conclude that liver tissue mimicking the hepatic plate structure contributes to the recovery and maintenance of hepatocyte polarity so that hepatocyte phenotype and differentiated functions in vitro will be much more resemble to the state in vivo. We are planning to construct human liver tissue mimicking the hepatic plate structure by means of microfluidic chip. Compared with the other culture patterns, we measure their hepatic functions to find a novel three-dimensional culture pattern in vitro and bioreactor that is proper to the application of bioartificial liver. Based on the above research, we can lay a solid foundation for bioartificial liver, treatment by transplantation of hepatocyte, and study of liver pathology and physiology.Methods:1. Microfluidic chip fabrication and design:Fabrication:Standard soft lithography and replica molding techniques.Material:Polydimethylsiloxane (PDMS)Cooperator:Shanghai Institute of Microsystem and Information Technology (SIMIT), Chinese Academy of Sciences (CAS),Design:As is shown in the article.2. Preparation of cells, solution and arrangement of experimental groups①Preparation of cells:human hepatocytes lines:C3A, HepGL, CL1(LO2)human endothelial cell line:EA.hy926culture medium:DMEM+10%FBS+1%Penicillin-Streptomycin②Preparation of solution:Sodium alginate solution+C3A:3×107 C3A/ml,0.7%(w/v)sodium alginate,0.9% NaCl,1% BSA,10mmol/l HEAPSSodium alginate solution+EA.hy926:1×107 EA.hy926/ml,0.7%(w/v) sodium alginate,0.9% NaCl,1%BSA,10mmol/l HEAPSGelation solution:10% dextran,20mmol/1BaC12、0.72% NaCl、10mmol/l HEAPS Buffer solution:10% dextran,0.9% NaCl, 10mmol/1 HEAPS③Arrangement of experimental groupsExperimental group:three-dimentional co-culture mimicking the hepatic plate structure (C3A:EA.hy926= 3:1)Control group 1:hybrid three-dimentional co-culture (C3A:EA.hy926= 3:1)Control group 2:hybrid monolayer co-culture (C3A:EA.hy926= 3:1)3. Operation of the above Experimental group and Control groups:Experimental group:These cell suspensions and buffer/gelation solutions were continuously introduced into the chip channel with syringe pumps. The flow rates of the C3 A suspension, EA.hy926 cell suspension, buffer solution, and gelation solution were 20ul/mim 10ul/min、15ul/min、4ml/h (66.7ul/min) respectively. Because the multiphase flows formed in the microchannel for cell suspensions and buffer/gelation solutions reach a stable laminar flow, the initial positions of the 2 cell types are maintained throughout the gelation process. Then the hepatic plate-incorporating alginate hydrogel fibers are formed. After that, the three experimental groups are cultured with complete medium in cultured in TissueFlex(?) microbioreactors(Oxford University). To find out the best flow rate and whether the hepatic plate-arrangement has formed, we utilize different color dyes and double-fluorescence staining.Control group 1:These two kinds of cells are mixed with each other. Then the hybrid cell suspensions and buffer/gelation solutions were continuously introduced into the chip channel with syringe pumps. The remaining steps are the same with Experimental group.Control group 2:These two kinds of cells are mixed with each other. Then they are cultured with complete medium in cultured in TissueFlex(?) microbioreactors(Oxford University).Recover the hepatic plate structure from the fibers by dipping the alginate fiber in PBS containing 1 unit/mL alginate lyase when the cell colonies show a stable hepatic plate-like morphology.4. Morphological observation and vitality and functional evaluation of hepatocytes①Cell morphologies:Inverted microscope②Histological observation:Double-fluorescence immunohistochemistry③Cell viabilities:CCK-8④Functional determination:Bilirubin, GCK, G6P, ALB, TF, AAT, APOA1, ALT, AST, LDH, ALP, CPS1, GS, Urea, NH3, CYP3A4, CYP2D6, CYP1A2, diazepam, GST, F2, F5, F7, PXR, TT.⑤RT-QPCR:UGT1A1, GCK, G6PC, ALB, TF, APOA1, AAT, ALT11, GOT2, LDHA, ALPL, CPS1, GLUL, CYP3A4, CYP2D6, CYP1A2, GSTA1, F2, F5, F7, PXR/NR1I2,β-actin.5. Statistical and outcome analysesAll values are reported as the mean±SD values on the basis of at least 3 individual samples. The significance of the differences between groups are tested by one-way analysis of variance (ANOVA) followed by either the Tukey or GamesHowell post-hoc test using SPSS Statistics 13 software. A probability value of P<0.05 is considered statistically significant.Results:1. Microfluidic chip fabrication and design:The Microfluidic chip we designed can be applied to construct human liver tissue mimicking the hepatic plate structure. It’s design is feasible.2. The operation of the Experimental group:These cell suspensions and buffer/gelation solutions were continuously introduced into the chip channel with syringe pumps. The flow rates of the C3A suspension, EA.hy926 cell suspension, buffer solution, and gelation solution were 20ul/min、10ul/min、15ul/min、4ml/h (66.7ul/min) respectively. Because the multiphase flows formed in the microchannel for cell suspensions and buffer/gelation solutions reach a stable laminar flow, the initial positions of the 2 cell types are maintained throughout the gelation process. Then the hepatic plate-incorporating alginate hydrogel fibers are formed. C3A are localized in the fiber core, whereas EA.hy926 are located on both sides of the hepatocytes. They are arranged into the hepatic plate structure. The average diameter of hydrogel microfibers is 100um.3. The outcome of different color dyes and double-fluorescence staining:By utilizing different color dyes, we come to a conclusion that the flow rates reach a stable laminar flow. And further double-fluorescence staining outcomes verify the fact that the hepatic plate-incorporating alginate hydrogel fibers are formed. C3A are aligned in 2 or 3 rows and localized in the fiber core, whereas EA.hy926 are located on both sides of the hepatocytes. They are arranged into the hepatic plate structure.4. Morphological observation of hepatocytes:At the beginning of the culture stage, hepatocytes in all of the groups have shown a tendency to proliferate. As time goes on, hepatocytes and endothelial cells in Experimental group and Control group 1 start to merge into a micro liver tissue at day 6 and 8 respectively. The cell proliferation is limited and reaches a stable maintenance stage. And the morphology of cells in Experimental group is better than that in Control group 1. Compared with them, hepatocytes in Control group 2 show a tendency to apoptosis as a result of the limited growing space and contact inhibition.Conclusion:We have achieved the goal of constructing human liver tissue mimicking the hepatic plate structure by means of microfluidic chip and established the technical system. Based on the microfluidic chip and the technology, we have developed a new three-dimensional culture mode and the hepatocyte phenotype and differentiated functions are likely to be much more resemble to the state in vivo. Becides, it lies a solid foundation for further research. What’s more, the application of human cells makes the research more applicable to bioartificial liver, treatment by transplantation of hepatocyte, and study of liver pathology and physiology.