Spheroid Formation and Expression of Liver-Specific Functions of Human Hepatocellular Carcinoma-Derived FLC-4 Cells Cultured in Lactose-Silk Fibroin Conjugate Sponges

被引:30
作者
Gotoh, Yohko [1 ]
Ishizuka, Yasuyuki [2 ]
Matsuura, Tomokazu [3 ]
Niimi, Shingo [4 ]
机构
[1] Natl Inst Agrobiol Sci, Silk Mat Res Unit, Tsukuba, Ibaraki 3058634, Japan
[2] Appl Cell Biotechnol Inc, Tsurumi Ku, Kanagawa 2300045, Japan
[3] Jikei Univ, Sch Med, Dept Lab Med, Minato Ku, Tokyo 1058461, Japan
[4] Natl Inst Hlth Sci, Div Biol Chem & Biol, Setagaya Ku, Tokyo 1588501, Japan
关键词
RAT HEPATOCYTE SPHEROIDS; SYNTHETIC EXTRACELLULAR MATRICES; HYBRID ARTIFICIAL LIVER; MESENCHYMAL STEM-CELLS; RADIAL-FLOW BIOREACTOR; IN-VITRO; BIOARTIFICIAL LIVER; DRUG-METABOLISM; BONE-MARROW; ENDOTHELIAL-CELLS;
D O I
10.1021/bm101495c
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 beta-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 mu m in diameter and elongated pores 250-450 mu m in length and 100-150 mu m 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 mu m 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-4 alpha. On the other band, 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.
引用
收藏
页码:1532 / 1539
页数:8
相关论文
共 55 条
[1]   Structural polarity and functional bile canaliculi in rat hepatocyte spheroids [J].
Abu-Absi, SF ;
Friend, JR ;
Hansen, LK ;
Hu, WS .
EXPERIMENTAL CELL RESEARCH, 2002, 274 (01) :56-67
[2]   Silk-based biomaterials [J].
Altman, GH ;
Diaz, F ;
Jakuba, C ;
Calabro, T ;
Horan, RL ;
Chen, JS ;
Lu, H ;
Richmond, J ;
Kaplan, DL .
BIOMATERIALS, 2003, 24 (03) :401-416
[3]  
Aoki H, 2003, BIO-MED MATER ENG, V13, P309
[4]   Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: Hepatocytes cultured in a sandwich configuration [J].
Berthiaume, F ;
Moghe, PV ;
Toner, M ;
Yarmush, ML .
FASEB JOURNAL, 1996, 10 (13) :1471-1484
[5]   COMPARISONS OF 2-INVITRO CYTO-TOXICITY ASSAYS - THE NEUTRAL RED (NR) AND TETRAZOLIUM MTT TESTS [J].
BORENFREUND, E ;
BABICH, H ;
MARTINALGUACIL, N .
TOXICOLOGY IN VITRO, 1988, 2 (01) :1-6
[6]   Hepatocyte cell lines:: their use, scope and limitations in drug metabolism studies [J].
Castell, Jose V. ;
Jover, Ramiro ;
Martinez-Jimenez, Celia P. ;
Gomez-Lechon, Maria Jose .
EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY, 2006, 2 (02) :183-212
[7]   Liver-enriched transcription factors and hepatocyte differentiation [J].
Cereghini, S .
FASEB JOURNAL, 1996, 10 (02) :267-282
[8]   Galactose-carrying polymers as extracellular matrices for liver tissue engineering [J].
Cho, CS ;
Seo, SJ ;
Park, IK ;
Kim, SH ;
Kim, TH ;
Hoshiba, T ;
Harada, I ;
Akaike, T .
BIOMATERIALS, 2006, 27 (04) :576-585
[9]  
CHOMCZYNSKI P, 1987, ANAL BIOCHEM, V162, P156, DOI 10.1016/0003-2697(87)90021-2
[10]   Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold [J].
Chua, KN ;
Lim, WS ;
Zhang, PC ;
Lu, HF ;
Wen, J ;
Ramakrishna, S ;
Leong, KW ;
Mao, HQ .
BIOMATERIALS, 2005, 26 (15) :2537-2547