title: Primary human hepatocytes from metabolic-disordered children recreate highly differentiated liver-tissue-like spheroids on alginate scaffolds.
abstract: Human hepatocyte transplantation has not been routinely established as an alternative to liver transplantation in liver disease due to low cell engraftment rates. Preimplantation in vitro engineering of liver tissue using primary human hepatocytes on three-dimensional scaffolds could be an alternative model. Alginate bioscaffolds were seeded with 1×10(6) hepatocytes freshly isolated from the livers of three children suffering from different metabolic disorders. During a culture period of 14 days only a marginal loss of hepatocytes was observed via measurement of DNA content per scaffold. Formation of hepatocyte spheroids was detected from day 3 onward using transmission light microscopy. Biochemical assays for albumin, α1-antitrypsin, and urea revealed excellent metabolic function with its maximum at day 7. Low lactate dehydrogenase enzyme release demonstrated minor cellular membrane damage. Hematoxylin and eosin and periodic acid Schiff staining displayed high cell viability and well-preserved glycogen storage until day 7. Immunofluorescent staining of hepatocyte nuclear factor 4, zonula occludens protein 1, and cytokeratin 18 revealed highly differentiated hepatocytes in spheroids with a tissue-like structure on scaffolds. Fluorescent labeling of cytochrome P450 and bile canaliculi demonstrated detoxification ability as well as a well-shaped bile canaliculi network. Almost constant expression levels in most target genes were detected by quantitative real-time polymerase chain reaction. The results of TUNEL reaction implicated a safe scaffold-dissolving procedure. Our results indicate that alginate scaffolds provide a favorable microenvironment for liver neo-tissue recreation and regeneration. Further, we demonstrate that livers from children with inherited metabolic disorders could serve as an alternative cell source for in vitro experiments.
authors.affiliation: Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany .
authors.forename: Jeanette
authors.initials: J
authors.lastname: Bierwolf
authors.forename: Marc
authors.initials: M
authors.lastname: Lutgehetmann
authors.forename: Steffen
authors.initials: S
authors.lastname: Deichmann
authors.forename: Johannes
authors.initials: J
authors.lastname: Erbes
authors.forename: Tassilo
authors.initials: T
authors.lastname: Volz
authors.forename: Maura
authors.initials: M
authors.lastname: Dandri
authors.forename: Smadar
authors.initials: S
authors.lastname: Cohen
authors.forename: Bjoern
authors.initials: B
authors.lastname: Nashan
authors.forename: Joerg-Matthias
authors.initials: JM
authors.lastname: Pollok
mesh_terms: D000464:Alginates / Q000494:pharmacology*; D001681:Biological Assay; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000473:pathology; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002675:Child, Preschool; D004247:DNA / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D020723:Glucuronic Acid / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000637:transplantation; D006603:Hexuronic Acids / Q000494:pharmacology; D006801:Humans; D007223:Infant; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology*; D008659:Metabolic Diseases / Q000473:pathology*; D010503:Periodic Acid-Schiff Reaction; D012709:Serum Albumin / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D054457:Tissue Scaffolds / Q000737:chemistry*; D014508:Urea / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D012709:Serum Albumin; D000515:alpha 1-Antitrypsin; D020723:Glucuronic Acid; D014508:Urea; D006003:Glycogen; D004247:DNA; D007770:L-Lactate Dehydrogenase