Preliminary Exploration Of Bioartificial Liver Neotype Biomaterial

Background and Objection:There is a high prevalence of hepatopathy in China. Acute liver failure, with a fatality rate up to 60 %-90%. is a serious clinical syndrome due to various hepatic diseases. Bioartificial liver (BAL) support system as an effective treatment of acute liver failure and an alternative to liver transplantation is being considered worldwide. Bioartificial liver is an extracorporeal device based on cultured hepatocytes, which can perform the main biological support roles of synthesis,detoxification, metabolism and other functions. Therefore, the liver cell source, as the biomaterial, is the core component of the BAL.Ideal cell source of the BAL would combine the following features: (1)Human-derived; (2) Normal Phenotype; (3) Easily accessible; (4) Easy to culture and enable to proliferate to a high density; (5) Well-differentiated and capable of the entire biological metabolism which the mature hepatocytes have. But all the cell sources so far used for the BAL do not completely fulfill all the criteria above. The source of the cellular component which is common used and their defects are as following: (1) Freshly isolated adult primary hepatocytes, which are capable of full amount of all the liver-specific functions, are no doubt the most ideal biomaterial of the BAL. However, the cell sources are in insufficient quantity and are difficult to proliferate in vitro, which make it difficult to fulfill the clinical popularization and applications. (2) Xenogenic liver cells, such as porcine hepatocytes. Several types of the BAL which have entered clinical trials use them as biomaterials. However, many European nations explicitly prohibit using porcine hepatocytes, which carry the potential of zoonosis and might introduce anaphylactic reactions,as the cell source of the BAL. (3) Human-derived cell lines, including tumor-derived cell lines and immortalized hepatocytes obtained from genetic engineering. Tumor-derived cell lines, which have extensive sources, are capable of some liver-specific functions,and can proliferate in culture with sufficient rate to the required amount of BAL treatment, but their potential risk of tumorigenesis can not be completely excluded at present. The immortalized hepatocytes were obtained by introduction of a simian virus large T antigen (SV40T) gene in to liver cells using a retroviral vector, but SV40LT also have inherent tumorigenic ability and the retroviral vector may lead to unpredictable genetic disorders. (4) Other cell sources, such as hepatic stem cells, are far from the stage of applying research,since the isolation and culture of stem cells as well as the technology of inducing them into hepatocytes are not yet mature.It yet difficult to be a reliable and effective approach of treatment due to the lack of promising biomaterials, however, researchers believe that the BAL will finally bring a revolutionary change to the treatment for acute liver failure as research continues. Based on previous researches, this study brings forward our own point of view and makes a preliminary exploration focusing on the security problems of human-derived cell lines. Firstly, targeting at tumor-derived cell lines, we propose to construct a neotype human-derived cell line from well-differentiated hepatocellular carcinoma or noncancerous liver tissues. Secondly, aiming at the immortalized hepatocytes obtained from genetic engineering,the study investgates the new carrier systems of immortalized hepatocytes based on the piggyBac. At last,we conduct a pilot study on the feasibility of nitrocellulose (NC) as a support material of culturing hepatocytes. In short, the present study makes efforts to investigate the crucial problem for the development of BAL treatment: the ideal biomaterials.Methods and materials1?Comparison of different human liver cell lines in liver function level.Surgical resection specimens of liver surrounding non-tumor tissue was collected in Nanfang Hospital and separated using our liver cell separation apparatus boxes which we have applied for a patent (Patent No.: 201120133192.5) . The primary hepatocytes were cultured in RPMI-1640 medium containing 15% fetal bovine serum, routine conditions.RNA from 12 different liver cell lines and primary hepatocytes was extracted and detected by real time PCR of liver functionally related genes mRNA expression. The functionally related protein ALB and CYP2E1 were detected by immunofluorescence, meanwhile ALB and BUN from culture supernatants were evaluated by automatic biochemical analyzer and the expression of the functional proteins were examined by immunofluorescence.2?Comparison of various differentiation degrees of liver tumors in liver function level. We collected 34 surgical resection specimens between January,2009 and December, 2011 in Nanfang Hospital which has been pathologically confirmed liver tumors of various differentiation degrees and the margin without tumor-infiltrating. The liver tumor specimens contain well-differentiated HCC tissues, poorly differentiated HCC tissues, and relatively normal liver cancer tissue of 12 cases respectively. The functionally related genes of the three types of tissue were detected by real time PCR and ALB and CYP2E1 expressions were examined by immunohistochemical.3?Establishment of neotype human-derived cell lines for the BAL. All procedures performed within above-mentioned primary hepatocyte culture. Cells were grown in medium (Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (FBS)) and digested in 0.25% trypsin. Expression of hepatocyte special functions, compared to normal adult hepatocyte and C3A cells,were evaluated via real-time PCR and supernatants assays.4?Construction and application of a vector for virus-free hepatocyte reversible immortalization. We obtained attB1-Kozak-eGFP /F2A/neo-attB2, attB1-Kozak-hTERT-attB2 and attBl-hTERT/E2A/eGFP/F2A/neo-attB2 through PCR amplification, then we recovery the product through gel extraction, and then we through Gateway, a recombinant clone technology including BR reaction and LR reaction, and endonuclease digestion and conjunction to construct the vector PB-hTERT/E2A/eGFP/F2A/neo. Gene expression analysis by real-time PCR for HL-7702 cells co-transfected by PB-hTERT/E2A/eGFP/F2A/neo and pCAGG-PBase were further conducted.5?The properties of nitrocellulose membrane as a scaffold for cells of BAL.Cells seeded on the NC membranes were examined by scanning electron microscope and HE staining,expression of special function marker by immunocytochemistry,aptosis by flow cytometry and Lactate dehydrogenase leakage(LDH) on nitrocellulose membrane. Cells cultured on coverslips or plates served as controls.6?Statistical Analysis: The difference of mRNA expression among the groups were analyzed by homogeneity test of variances, and K-independent samples nonparametric tests were used under an equal condition; Count data was analyzed using chi-square test; The differences of leakage rate of lactate dehydrogenase (LDH)between groups was analyzed using two-way ANOVA; Differences between groups of apoptosis detection are compared by two independent T test. The level of significance was defined as P<0.05. All statistical tests were performed using SPSS software version 13.0.Results:1?Comparative study of hepatocyte function among different human liver cell lines. Gene expressions of 12 liver special functions were much lower than which of normal adult hepatocyte. The difference of mRNA expression detected by Real time PCR of liver functional genes among the liver cell lines was huge. The specific liver function of HepG2, C3A and Hep3B2.1-7 were relatively better, especially the synthesis function. Cell immunofluorescence confirmed the protein expression of partial correlation genes, the content of ALB and BUN from culture supernatants were similar to the results of real time PCR.2?Comparative study of hepatocyte function among hepatocellular carcinoma with different differentiation. The difference in expression of 12 hepatocyte special gene related to liver synthesis and metabolism as well as detoxification via real time PCR, excepting GST-?, were statistically significant (?2 ?16. 635, P<0.001).Adjacent tissues exhibited best expression of liver special functions compared with well-differentiation and poor-differentiation tissues. Expression of ALB and CYP2E1 by Immunohistochemical assays were similar to above-mentioned PCR results.3?Establishment of neotype human-derived cell lines for the BAL. Cell lines from well-differentiated hepatocellular carcinoma and noncancerous liver tissues were established, and were named NHBL1 and NHBL2 respectively. The expression of 12 hepatocyte special gene of the NHBL1 were evaluated via real-time PCR, and GST-? was slightly higher while others were lower at different levels than C3A cells.The differences of AAT, TF and G6P were less than one times. The difference of ALB, TTR and CYP 3A4 were less than two times. The difference of CPS-1 and CYP3 A were less than five times. The difference of other genes were more than five times. The productions of ALB for single cell in Culture supernatant were 2.200×10-5g/L, 0.277×10-5g/L, 0.496×10-5g/L respectively, while the productions of BUN were 1.965×10-5mmol/L 0.109×10-5mmol/L, 0.237×10-5mmol/L respectively.The NHBL2 derived from the nontumorous part of liver tissue of a 36 years old male patient with liver carcinoma, has been passaged more than 25 times (to date). In expression of 12 hepatocyte special gene via real time PCR, ALT, AAT, TF, TTR,TAT and CYP3A4 were slightly lower while others were higher at different levels than C3A cells.4?Construction and application of a vector for virus-free hepatocyte reversible immortalization. Successful construction of immortalized hepatocellular vector PB-hTERT/E2A/eGFP/F2A/neo, whose target sequence should be the same as GenBank's, was confirmed by PCR, restriction enzvme digestion and gene sequencing. We succeeded in co-transfecting HL-7702 cells with PB-hTERT/E2A/eGFP/F2A/neo and pCAGG-PBase.5?The properties of nitrocellulose membrane as a scaffold for cell of BAL. The morphology of nitrocellulose membrane, characterized by scanning electron microscope,showed smooth surface at low magnification and cavernous three-dimensional structure, high accuracy and consistency of pores diameter at high magnification. Cells were observed through HE staining and transparent treatment of membranes for both membrane and coverslip cultures. The cells gradually proliferated on membranes and exhibited similar morphology in light microscope and scanning electron microscope, normal expression of special function markers by immunocytochemistry, as well as similar aptosis to coverslips'(F=0.267,P=0.609).Conclusion1?The current human-derive liver cell lines still have greater disparity with ideal BAL biomaterials. There are many safety concerns about the cell lines derived from high malignant tumor tissues. In addition, human-derive liver cell lines still can't achieve the functional level of primary normal adult hepatocyte and can't satisfy the condition of ideal BAL biomaterial.2?Cell lines from well-differentiated hepatocellular carcinoma and noncancerous liver tissues are established, the functional level of which are similar to that of C3A cells. These mitigate the security concerns to a certain extent, and provide a new idea and means for establishing neotype cell sources for BAL.3?Construction and application of a vector for virus-free hepatocyte reversible immortalization based on piggyBac carrier system may became another more perfect way to solve the deficiency of ideal BAL.4?In this study we initially investigate the feasibility of nitrocellulose membrane as the scaffold material supported the BAL cell growth; the results demonstrated that nitrocellulose membrane may become a membrane material of BAL bioreactor.