| Background and ObjectionLiver failure,which can't be effectively improved by conventional medical treatment,is a severe clinical syndrome.The mortality of liver failure is up to 70 percent.Liver transplantion is the only effective treatment for hepatic failure,However,its application is limited by donor organ availability,there are less than 1%patients with liver failure can receive liver transplantation.The emergence of bioartificial liver(BAL)brought a glimmer of hope for patients with liver failure.It can partly replace diseased liver to gain time for hepatocyte regeneration,and it aslo can provide time to find a suitable donor liver.However,the lack of the ideal liver cell sources and efficient bioreactors seriously restricted the development of this emerging treatment technology.At present,only porcine hepatocytes and liver cancer cell lines C3A had entered clinical trials.Porcine hepatocytes may lead to immune rejection and cause zoonotic diseases as heterogonous cells;C3A was generated from high grade malignancy with a potential risk of tumorigenicity.Due to the shortcomings of existing biological materials and the lack of ideal biological materials,there were no official ratified bioartificial liver cell products in the world,and no any cell completely to meet the requisition of ideal biological materials for BAL.Then,the research of BAL got into the trough in recent years.Therefore,the difficulty and most important prerequisite of BAL development was to establish suitable hepatocyte sources,giving full play to the liver functions of synthesis,metabolism,detoxification.In recent years,scholars from various countries constantly explored new cell material sources using different methods.For instance,constructing liver cell lines by conveying SV40LT or hTERT gene into hepatocytes using virus vectors;establishing a new type of tumor-derived liver cell lines;imitating the hepatocyte microenvironment for growth,in order to improve the functional activity of hepatocytes.However,these technologies and products existed some shortcomings and deficiencies,and some of the products were on foreign enjoyment of patent protection.Therefore,the research of new BAL materials has been constantly supported by the national 863 Program.Our group also detected liver function of 12 liver-derived cell lines,and found that the level of liver function of existing hepatocyte lines were significantly lower than the normal primary hepatocytes,which can't satisfy the requisition of BAL.In preliminary studies,we also planned to construct a high-functioning liver adenoma cell lines using hepatic benign hepatic adenoma tissue,but it was difficult to obtain fresh surgical specimens because of the very low incidence of hepatic adenoma and difficult to diagnose before surgery.Our group further detected liver-derived tumor tissues with different degree of differentiation,and found that well-differentiation exhibited better expression of liver special functions than poor-differentiation tissues,but all lower than adjacent normal liver tissues.Based on this preliminary work,allowing for both security and functionality,we carried out the construction of well-differentiated hepatocellular carcinoma cell lines.Eventually,we successfully established a hepatoma cell line designated NHBL2 from 36-year-old adult well-differentiated HCC tissues.In order to evaluate of the value of its application in BAL,we further detected their biological characteristics and functional level,and tried to cultivate it in hydrogel.Methods and Materials1.Isolation and culture of human primary hepatocytesSurgical resection specimens of well-differentiated HCC tissues were collected in Nanfang Hospital,and primary hepatocytes were separated using the two-step collagenase perfusion for small piece of liver tissue established in our laboratory,which were approved by the Medical Ethics Committee,and obtained the consent of the patients and their families.2.Subcultivation,cryopreservation and recovery of NHBL2 cell linesRemoving fibroblast-like cells from primary cultured liver cell using selective digestion and repeated attachment method,epithelioid cells gradually were purified and obtained growth advantage with gradually disappearing of a small amount of fibroblasts in the training process.In order to ensure the hepatocytes maintaining high levels of liver cell activity,detected biological characteristics of the cells regularly,discarded the hepatocytes with weak specific liver functions and low proliferative capacity,and continued to cultivate the hepatocytes with well specific functions and high proliferative capacity in the continuous cells subcultivation.3.Establish cell lines with long-term SubcultivationNew human-derived hepatocytes lines consecutive passages over 50 generations in vitro,and cell morphology and the function markers were stable and not change significantly by three times detection.4.Biological characteristics of novel human hepatocyte lines1).Morphological observation:Changes in cell volume and morphology were observed under optical microscope.Ultrastructures of NHBL2 were observed under transmission electron microscope.2).The proliferation kinetics detect:drawing cell growth curveby cell counting,detecting the cell cycle by flow cytometry..3).Function detection:?Detection of hepatocytes-related function in gene level:mRNA expression of NHBL2 cell line was detected by qRT-PCR.?Detection of hepatocyte-related function in protein level:Western-blot,immunocytochemistry and immunofluorescence were used to detect the expression of important functional protein and surface markers of NHBL2 cell line;PAS stain was performed to measure the glycogen synthesis and storage capacity of NHBL2 cell line;albumin and urea nitrogen content in culture supernatant of NHBL2 cell line was detected by Automatic biochemical analyzer.?Function optimization experiments:After cultured in hydrogel for one week,mRNA of NHBL2 cell line was extracted and detected the changes of hepatocytes functional gene expression by qRT-PCR.4).Safety evaluation:HBs-Ag?HBV-DNA and HCV-DNA content in culture supernatant of NHBL2 cell line was detected.5).Genetic characteristics detection:?Genomics detection:The whole gene structure of NHBL2 cell line was detected and compared with C3Acell line.?Karyotype analysis was performed to detect if there are any chromosomal mutation and ectopic in NHBL2 cell line or not.Results1.The Isolated primary hepatocytes gained larger and polygonal morphology in adherent culture,mixed with a few long-spindle interstitial cells.The isolated hepatocytes were gradually purified after being subcultured.They finally revealed an irregular polygonal cell morphology and appeared smaller than primary hepatocytes after the successful construction of NHBL2 cells.2.Proliferation kinetics:The cell growth curve made by cell counting showed that NHBL2 cells began to enter the fast-growing period 72 hours after adherent to the culture flasks.The cell doubling time of NHBL2 cell line was about 27 hours..At the same time,the cell cycle measured by flow cytometry proved that the proportion of NHBL2 in S + G2/M phase was slightly smaller(.335)than that of C3A cells in S + G2/M phase(0.379),but there was no statistical difference between two groups(p = 0.759,n = 3).3.Function testing:The mRNA expression levels of relative liver function markers in NHBL2 suggested that all parameter are slightly lower than the C3A cell line except for the glucose-6-phosphate which was slightly higher than the C3A cell line.This difference was rather small,around 1-5 times between the two.Semi-quantitative and quantitative test in the two cell lines showed the results substantially coincides with the level of gene expression.Function optimization experiments:After cultured in hydrogel for one week,hepatocytes-related function of NHBL2 cell line in gene level was improved.4.Safety evaluation and detection of genetic characteristics:.The content of HBs-Ag,HBV-DNA and HCV-DNA in NHBL2 cell line culture supernatant were all within the normal range.Karyotype analysis results suggested most of NHBL2 cells are polyploid and part of chromosomes were mutated and ectopic.According to genomic analysis,there was a certain similarity in the composition between the genes of NHBL2 and C3 A while discrepancy in gene composition still exists.ConclusionIn this study,we constructed a new liver cell line named NHBL2,derived from well-differentiated hepatocellular carcinoma,which was similar to C3A cell line in aspect of liver functions,but better than C3A in aspect of security,and also we cultivated NHBL2 cells in hydrogel to improve the functional activity,providing a new way of thinking for exploring new BAL biological materials. |
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