Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes

Use of hepatocytes in developing hybrid artificial liver is a topic of activeresearch . however,hepatocytes primary cultures remain viable for only a few weeks under normal cultivation使用adenovirus vector and introducing SV40 primary gene into rat and marmoset conditions. By using adenovirus vector and introducing SV40 primary gene into rat and marmosethepatocytes的primary cultures .我已经在转换转换中得到了支持cultivation period, immortalization, and massive cultivation. By using the same techniqueI have attempted to produce hybrid artificial liver by using human hepatocytes.Methods and Results：1. Adenovirus vector produced by recombination of E1A and E1B genes in human Adenovirus withmultiple deletion SV40 primary geneswas added to human primary cultures of hepatocytes by MOI (multiplicity of infection) and cultivated37℃for two小时，then fixed in ethanol after 48小时，additional cultivation与immunostained by using antibody to SV40T antigen. Rate of T antigen positive was apprMore oximately 20% in MOI 100, 4% in MOI 10, less than 0.5% in MOI 12. Adenovirus vector was introduced in human hepatocytes in MOI100 and plated to 1xl0 i D16 i D1 flask(25 cm i D13 i D1). After 3-4 weeks，approximately 100/colonies undergoing transformed conversion were produced from 10 cells.3. Humanhepatocytes introducing with primary SV40 genes were cultivated as a bulk for longer than 12 monthsand the cells mostly SV40T antigen positive，continued to reproducefavorably and attained immortalization. Also immunostaining with albumin waslocalization in cytosol of these cells.Conclusion：使用adenovirus vector efficient introduction of SV40 primary gene and transformed conversionwas possible resulting in massive cultivation and immortalization.discussion:Under the single layer cultivation method work this time，hepatocyte function was found to deterorated with time by measuring urea production and tyrosineamino transferase (TAT) activity. I plan to used third degree high dense cultivation to improve thisaspect in the future. Less

Use of primary culture of hepatocytes in developing hybrid artificial liver is a topic of active research. However, primary cultures of hepatocytes remain viable for only a few weeks under normal cultivation conditions. By using adenovirus vector and introducing SV40 primary gene into rat and marmoset primary cultures of hepatocytes. I have succeeded in transformed conversion resulting in longer cultivation period, immortalization, and massive cultivation. By using the same technique, I have attempted to produce hybrid artificial liver by using human hepatocytes.Methods and Results: 1. Adenovirus vector produced by recombination of E1A and E1B genes in human adenovirus with multiple deletion SV40 primary genes, was added to human primary cultures of hepatocytes by MOI (multiplicity of infection) and cultivated under 37℃ for two hours, then fixed in ethanol after 48 hours of additional cultivation, and immunostained by using antibody to SV40T antigen. Rate of T antigen positive was appr … More oximately 20% in MOI 100, 4% in MOI 10, less than 0.5% in MOI 1, and these results were depend on MOI.2. Adenovirus vector was introduced in human hepatocytes in MOI 100 and plated to 1xl0ィイD16ィエD1 flask(25 cmィイD13ィエD1). After 3-4 weeks, approximately 100/colonies undergoing transformed conversion were produced from 10 cells.3. Human hepatocytes introducing with primary SV40 genes were cultivated as a bulk for longer than 12 months and the cells mostly SV40T antigen positive, continued to reproducefavorably and attained immortalization. Also immunostaining with albumin was localization in cytosol of these cells.Conclusion: By using adenovirus vector efficient introduction of SV40 primary gene and transformed conversion was possible resulting in massive cultivation and immortalization.Discussion: Under the single layer cultivation method employed this time, hepatocyte function was found to deterorated with time by measuring urea production and tyrosine amino transferase (TAT) activity. I plan to used third degree high dense cultivation to improve this aspect in the future. Less