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.

Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells

“…Such a 3D culture model has occurred to recapitulate many in vivo tissue structures and functions [3,9]. Very few hepatocyte spheroid models were established using: a poly-(L-lactic acid ) polymer [10], rock techniques [11], micro-rotation flows [12], alginate scaffolds [13], RGD and galactose-conjugated membranes [14], positive-charged substrates [4], micropatterning techniques [15], nanopillar sheets [16], galactosylated nanofiber scaffold [17], or polyurethane forms [18]. However, hepatocyte spheroids under the influence of fetal calf serum and nonparechyalmal cells have not yet been established.…”

Role of Biological Scaffolds, Hydro Gels and Stem Cells in Tissue Regeneration Therapy

“…3 But human hepatocyte transplantation is also used as alternative to liver transplantation. 57 ( Figure 5). By using microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices which provide tissue-specific microenvironments for efficient tissue regeneration.…”

Liver tissue engineering using functional marine biomaterials

“…Furthermore, they cultured newborn rat liver cell isolates consisted of proliferating hepatocytes and progenitors into macroporous alginate MCs (pore size of 50-100 μm) to induce differentiation and maturation of the cells into functional hepatic tissue (Dvir-Ginzberg et al, 2008). Bierwolf et al (2012) seeded primary human hepatocytes isolated from the liver of metabolic-disordered children to recreate differentiated liver tissue-like spheroids on alginate MCs. Khattak et al (2007) investigated effect of perfluorocarbons (PFCs) as oxygen carrier on metabolic activity and viability of HepG2 cells encapsulated into alginate MCs because the growth of cells after encapsulation is hindered by insufficient oxygen supply.…”