Establishment and characterization of immortalized porcine neonatal hepatocytes without the use of viral components

Porcine hepatocytes are a promising option for xenotransplantation in light of the critical shortage of orthotopic donor livers. Because primary hepatocytes have limited ability to proliferate in vitro, several immortalized hepatocyte lines have been established. However, these cells have typically...

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Bibliographic Details
Published in:In vitro cellular & developmental biology. Animal 2020-01, Vol.56 (1), p.75-84
Main Authors: Zou, Yunlong, Hao, Haiyang, Li, Ning, Li, Qiuyan
Format: Article
Language:English
Subjects:
Animal Genetics and Genomics
Biomedical and Life Sciences
Cell Biology
Cell Culture
Developmental Biology
Electroporation
ESTABLISHMENT OF CELL LINES
Gene expression
Glycogen
Glycogens
Growth rate
Hepatocytes
Life Sciences
Lipids
Liver diseases
Medical treatment
Morphology
Neonates
Pathogenesis
Polymerase chain reaction
Serum albumin
Stem Cells
Telomerase reverse transcriptase
Therapeutic applications
Transfection
Viruses
Xenografts
Xenotransplantation
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Summary:Porcine hepatocytes are a promising option for xenotransplantation in light of the critical shortage of orthotopic donor livers. Because primary hepatocytes have limited ability to proliferate in vitro, several immortalized hepatocyte lines have been established. However, these cells have typically been generated using a virus-dependent transfection methodology and express viral oncogenes that introduce potential risks in clinical applications. In our study, we established immortalized porcine neonatal hepatocytes by introduction of a plasmid-based hTERT gene expression system by electroporation, without the use of viral components. We detected stable expression of hTERT by RT-PCR and Western blot. The immortalized hepatocytes exhibit a high growth rate, but retain the normal morphology of freshly isolated primary hepatocytes. To date, these immortalized hepatocytes have been expanded for over 80 passages. In addition, no significant differences were detected in glycogen synthesis, secretion of serum albumin, or lipid accumulation between the primary hepatocytes and our immortalized hepatocytes. The cells also exhibit serum-dependent growth and have no capacity for anchorage-independent growth in vitro, demonstrating that they have not been transformed in vitro. Our immortalized porcine hepatocytes will be useful for elucidating the pathogenesis of liver disease and developing efficient treatments. Furthermore, these immortalized hepatocytes may provide a safer source of cells for application in xenotransplantation, compared with immortalized cells generated using viral components.
ISSN:1071-2690
1543-706X
DOI:10.1007/s11626-019-00407-7