Construction Of In Vitro Liver Tissue Model Based On 3D Bioprinting Technology And Its Function Researc

BackgroundThe treatment of both primary and metastatic liver cancers has improved dramatically over the past few decades.However,a quantitative analysis of the numerous high-impact literature in this field has not yet been performed.In this paper,we take hepatocellular carcinoma and colorectal cancer liver metastases as the main research objects,and conduct a comprehensive summary and visual analysis of the literature in the therapeutic field of primary and metastatic liver cancer based on bibliometric analysis.We comprehensively demonstrate the development trend of treatment models,explore the shortcomings of current research and future development directions.MethodsWe performed a comprehensive search of the Clarivate Analytics Web of Science Core Collection database using search terms related to the treatment of hepatocellular carcinoma and colorectal liver metastases.We downloaded and analysed bibliographic information for relevant literature and explored research topics to understand their trends over time.We mainly use R software and the JavaScript-based EChart.js package for data analysis and visualization.ResultsFor research in the field of hepatocellular carcinoma therapy,we identified the 100 most-cited articles in the field as high-impact articles,with an average of 738 citations(range:349-6799).Most of the top 100 articles were from the United States(n=35),followed by Italy(n=28)and mainland China(n=26).Surgical treatment was the most studied topic(n=33).The late 1990s saw an increase in the number of high-impact articles,as did reports focusing on topical treatments.Publications starting in the 2000s have seen highly cited papers on targeted therapy and immunotherapy,while the number of publications on systemic therapy has increased.For research in the field of colorectal cancer liver metastases treatment,we identified 3436 studies related to this field.The total number of citations for these articles ranges from 0-2352,with publication years spanning 1976-2020.The United States,Japan and France published the most articles.The research topics of these articles include systemic chemotherapy,molecularly targeted therapy,liver resection,prognosis prediction,hepatic artery perfusion,radiofrequency ablation,and two-stage liver resection.ConclusionsBased on bibliometric analysis,we identified the most influential articles related to the treatment of primary and metastatic liver cancer to explore trends in their treatment modalities.We believe that the development of systemic therapy is the current research hotspot for liver cancer,especially molecular targeted therapy and immunotherapy.In addition,advances in surgical techniques,especially in liver transplantation,are bound to further improve the prognosis of patients with liver cancer.However,due to the lack of reliable preclinical models,many clinical trial drugs fail to achieve the expected research results.In addition,the growing organ shortage problem greatly limits the application of liver transplantation.Therefore,future research should focus on solving the above problems encountered in the current development.In the second part,we will discuss an emerging 3D bioprinting technology that can be used to build in vitro tissue models with great potential applications in disease modeling,organ repair and reconstruction.BackgroundIn the first part,we summarize the important development trends in the field of liver cancer treatment in recent decades.We believe that the lack of specific preclinical models has affected the translation of the latest drugs for liver cancer into the clinic,and that organ shortages have limited the development of liver transplantation.Three-dimensional bioprinting is an emerging technology that can be used to build in vitro tissue models.Therefore,we aimed to summarize the latest research progress of 3D bioprinting technology in liver-related fields to explore the application prospects of this technology in the field of liver.MethodsBased on the Clarivate Analytics Web of Science core collection database,we have provided a comprehensive summary and visual analysis of all literature related to the field of 3D bioprinted livers.ResultsIn total,we retrieved 71 articles related to 3D bioprinting in the field of liver.The earliest study was published in 2005,and the article with the highest total citations(TC=565)was published in 2014.The country with the most publications in this field is the United States(n=27),followed by South Korea(n=22)and China(n=16).The research topics of articles in this field can be roughly divided into three categories,namely focusing on printing technology(n=29,40.8%),bioinks(n=19,26.8%),and biomedical applications(n=23,32.4%).ConclusionsThree-dimensional bioprinting technology has initially shown great application potential in the construction of in vitro tissue models.However,in general,the research of this technology in the field of liver is still in its infancy,and there are still many problems to be solved,such as the application of primary cells,the improvement of printing technology,and the optimization of bio-inks.In the third part,we will use primary mouse hepatocytes to construct artificial liver tissue based on 3D bioprinting technology and conduct in-depth research on its liver function.BackgroundIn recent decades,the global burden of liver disease has been increasing year by year,and various liver diseases such as viral hepatitis,liver cirrhosis,liver cancer,non-alcoholic fatty liver disease,alcoholic liver disease,and drug-induced liver injury have seriously threatened human health.Due to the lack of specific research models,many drugs fail in translation to the clinic.In addition,liver transplantation is an important means for the treatment of various end-stage liver diseases,however,its application is limited by the shortage of organs,and cell therapy and tissue-engineered liver are potential alternatives.The 3D bioprinting technology that has emerged in recent years has shown great application value in the construction of in vitro tissue-organ models,and a variety of artificial organs or tissues have been successfully constructed.In this study,based on 3D bioprinting technology,we constructed liver tissue using primary mouse hepatocytes and conducted an in-depth study of its liver function.MethodsWe isolated and extracted primary hepatocytes from C57BL/6J mice,and achieved long-term stable expansion of primary mouse hepatocytes in vitro by modifying the culture medium.Then,based on 3D bioprinting technology,two bioink systems of gelatin/sodium alginate and GelMA/LAP were used to construct liver tissue.The activity of cells in 3D printed liver tissue was judged by morphological characteristics,cell survival staining and cell proliferation experiments.The in vitro function of 3D printed liver tissue was explored by immunofluorescence staining,qPCR,CYP induction experiments,PAS staining,DiL-ac-LDL staining,ICG uptake and excretion and other experiments.After that,the 3D printed liver tissue was transplanted into the abdominal cavity of mice,and a mouse model of acute liver failure was created by extreme hepatectomy.The overall survival time of the mice in the experimental group and the control group was observed,the serum liver function of the mice was detected,and the transplanted body was observed.Pathological morphological features and single-cell analysis.ResultsWe successfully constructed gridded 3D-printed liver tissue,with more than 95%of the cells still viable within 2 weeks of printing.Like homeostasis in vivo,3D-printed liver tissue cells proliferated slowly.qPCR experiments showed that compared with cells cultured in 2D and 3D sandwich cultures,3D printed liver tissues exhibited higher levels of expression of liver-specific genes,such as ALB,AAT,etc.Immunofluorescence staining experiments confirmed the expression of liver-specific proteins.CYP drug induction experiments showed that CYP450 enzymes such as CYP1A2,CYP1B1,CYP2A6 and CYP2E1 showed higher expression levels after drug induction.In addition,the 3D-printed liver tissue possesses basic liver functions such as glycogen storage,low-density lipoprotein uptake,and ICG uptake and excretion.Animal experiments showed that the transplanted 3D-printed liver tissue successfully obtained blood supply to maintain cell survival,and the transplantation of 3D-printed liver tissue containing hepatocytes significantly prolonged the survival of mice with acute liver failure,and serum liver function tests showed better trend.In addition,single-cell sequencing revealed that the transplanted 3D-printed liver tissue contained multiple cell types.ConclusionsPrimary mouse hepatocytes in liver tissue constructed based on 3D bioprinting technology can survive for a long time and have various liver functions such as protein synthesis,drug metabolism,substance uptake and storage.Transplantation experiments can prolong the survival of mice with acute liver failure,showing potential application value in the field of organ repair and reconstruction.