Construction Of Liver Tissue Based On 3D Printing Technology And The Activity Analysis In Vivo And Vitro

PART ONE Isolation and culture of rabbit primary hepatocytes using simple collagenase digestion methodObjective:To explore isolation and culture of primary hepatocytes from newborn rabbit by a simple and eco-nomical method of collagenase digestion.Methods:Rabbit primary hepatocytes were extracted from liver tissue pieces of new-born rabbit by 0.1%collagenase type IV digestion,and were cultured in vitro.Then the cells number,cell viability,and cell pro-liferation ability were detected.Results:The primary hepatocytes were round-like,neat rows,bright luster,with good stereo sense,and clear cell lines.1.12×106 hepatocytes could be extracted from 1 g liver,and survival rate of the hepatocytes was 77%.Cell proliferation capacity showed a tendency of rising first and falling later,and cell growth curve corresponds to general rule of primary cells growth.Conclusion:The method is simple,easy to operate,and no need special equipment,which is suitable for general laboratory to carry out extraction,isolation,and culture of primary hepatocytes.PART TWOConstruction of multilayered hydrogel structure of rabbit primary hepatocytes using 3D bioprinting techniqueObjective:To construct three dimensional(3D)structure lamella containing rabbit primary hepatocytes/alginate/gelatin blend using 3D bioprinting technique,so as to investigate the feasibility of 3D biological printing technique for the construction of structural fragments of liver like hydrogels.Methods:Rabbit primary hepatocytes were extracted from liver tissue pieces of newborn rabbit by collagenase type IV digestion.Alginate and gelatin were mixed to form hydrosol,and rabbit primary hepatocytes were added into the hydrosol.3D bioprinting system was used to construct 3D printed hepatic structures using rabbit primary hepatocytes/alginate/gelatin blend.The hepatic hydrogel structures were cultured,and the cells' survival rate was detected by live-dead cell double fluorescencestaining.Results:The 3D structures with the grid hydrogel composed of multilayerstaggered cylindrical microfilament were obtained.The size of the structure was about 10 mm×10 mm×1.2 mm,and pore size was about 450?m×450?m.The printed rabbit liver cells can still proliferate in the three-dimensional structure,the live/dead cell fluorescence staining results showed that the hepatocytes' survival rate was about 82%± 3%after printing,and the primary hepatocytes of rabbit were evenly distributed in thestructure.Conclusion:3D bioprinting of hepatocytes is feasible,which can provide a new way to construct liver-like tissue.PART THREELiver Fibrosis Model in Rabbit Induced by Subcutaneous Injection of Carbon Tetrachloride Objective:To use subcutaneous injection of carbon tetrachloride(CC14)to induce hepatic fibrosis model in rabbit.Methods:36 New Zealand white rabbits were divided into the experimental group(n=30)and the control group(n=6)randomly.The rabbits of experimental group were treated by subcutaneous injection of 50%CCl4 0.3 mL/kg in olive oil twice a week for the initial two weeks,then were treated by subcutaneous injection of 50%CCl4 0.2 mL/kg in olive oil twice a week for the next 10 weeks.The rabbits of control group were treated by same amount of olive oil.Rabbits' liver function,biochemical indicators,liver gross,histopathological changes and hepatic fibrosis degree were detected in experimental group and control group at 4th,8th,and 12th week respectively.Results:6 rabbits were dead in experimental group during the period of molding,and animal mortality rate was 20%.No dead case was found in control group.In experimental group,rabbits' liver fibrosis degree increased gradually with molding time extension.Serum alanine aminotransferase,aspertate aminotransferase,and gamma glutamyl transpeptidase increased significantly,while their liver tissue appeared false flocculus structure,and molding success rate was 80%.The liver function and histological structure of control group were normal.Conclusions:Injecting CCl4 for a long time may lead to the formation of liver fibrosis in rabbits,with significant difference in different time and high success rate.The model can be used further in related experiments.PART FOURStudy on 3D bioprinted liver structure implanting hepatic fibrosis's rabbit Objective:Three dimensional(3D)bioprinted liver-like hydrogel structures were implanted into rabbit liver fibrosis model by the liver surface cladding method to discuss its feasibility of improving liver fibrosis and generating liver-like tissue.Methods:Primary hepatocytes were extracted from liver tissue of rabbit by 0.1%collagenase type IV digestion,and hepatic fibrosis model was established by subcutaneous injection of carbon tetrachloride(CCl4)into rabbits.3D structure lamella containing rabbit primary hepatocytes/alginate/gelatin blend was constructed using 3D bioprinting technique.Then the 3D structure lamellas were implanted in the liver surface of hepatic fibrosis rabbits.At 16 days after implantation,the liver functional biochemical index and liver histopathological changes were examined,and the developmental degrees of hepatic fibrosis were observed as well as the formation of liver-like tissue structure.Results:Most of the each implant was not degraded at 16 days after implantation of rabbit hepatocytes hydrogel scaffold.The undegraded implant was closely integrated in the liver.By the biochemical indexes detection and liver tissue histopathological observation,the results showed that the fibrosis degree of the rabbits' liver and biochemical index of liver function had some improvements in experimental group.However,the comparisons between groups were P>0.05 and no statistical significance.Results of histological observation showed that hepatocytes were evenly distributed,and that cell degeneration and death were not observed,succeeding in generating liver-like tissue structure in the implant of experimental group.Conclusion:Bioprinted hepatic structure has the potential to regenerate liver-like tissue structure and rebuild liver functions in some extent.Our results demonstrated it's possible to build a bridge between basic medicine and clinical practice,and lay the foundation for the hepatic regeneration.