Primary human hepatocytes from metabolic-disordered children recreate highly differentiated liver-tissue-like spheroids on alginate scaffolds.
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Primary human hepatocytes from metabolic-disordered children recreate highly differentiated liver-tissue-like spheroids on alginate scaffolds. / Bierwolf, Jeanette; Lütgehetmann, Marc; Deichmann, Steffen; Erbes, Johannes; Volz, Tassilo; Dandri-Petersen, Maura; Cohen, Smadar; Nashan, Björn; Pollok, Jörg-Matthias.
in: TISSUE ENG PT A, Jahrgang 18, Nr. 13-14, 13-14, 2012, S. 1443-1453.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Primary human hepatocytes from metabolic-disordered children recreate highly differentiated liver-tissue-like spheroids on alginate scaffolds.
AU - Bierwolf, Jeanette
AU - Lütgehetmann, Marc
AU - Deichmann, Steffen
AU - Erbes, Johannes
AU - Volz, Tassilo
AU - Dandri-Petersen, Maura
AU - Cohen, Smadar
AU - Nashan, Björn
AU - Pollok, Jörg-Matthias
PY - 2012
Y1 - 2012
N2 - 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, ?1-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.
AB - 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, ?1-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.
M3 - SCORING: Journal article
VL - 18
SP - 1443
EP - 1453
JO - TISSUE ENG PT A
JF - TISSUE ENG PT A
SN - 1937-3341
IS - 13-14
M1 - 13-14
ER -