title: Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges.
abstract: This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.
authors: Silk-Materials Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. gotohy@affrc.go.jp
authors: Yohko
authors: Y
authors: Gotoh
authors: Yasuyuki
authors: Y
authors: Ishizuka
authors: Tomokazu
authors: T
authors: Matsuura
authors: Shingo
authors: S
authors: Niimi
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005349:Fibroins / Q000378:metabolism*; D006801:Humans; D007785:Lactose / Q000378:metabolism*; D008113:Liver Neoplasms / Q000473:pathology*; D047011:Silk / Q000378:metabolism*
chemical_list: D047011:Silk; D005349:Fibroins; D007785:Lactose