title: Three-dimensional co-culture of rat hepatocyte spheroids and NIH/3T3 fibroblasts enhances hepatocyte functional maintenance.
abstract: Functional maintenance of primary hepatocytes in culture can be improved by several distinct approaches involving optimization of the extracellular matrix microenvironment, media composition and cell-cell interactions, both homotypic and heterotypic. Using a galactose-decorated surface, we have developed a method to combine these two approaches by co-culturing rat primary hepatocyte spheroids with NIH/3T3 mouse fibroblast cells. Spheroids were performed by culturing hepatocytes for 3 days on galactosylated poly(vinylidene difluoride) membrane; NIH/3T3 cells were subsequently seeded and co-cultured with the spheroids. Results showed that although NIH/3T3 cells alone responded poorly to the galactosylated PVDF surface and displayed limited attachment, NIH/3T3 fibroblasts attached to the periphery of the hepatocyte spheroids and proliferated around them. Co-cultured hepatocyte spheroids exhibited significantly higher liver-specific functions as compared to spheroids cultured alone. Albumin secretion level in this co-culture system peaked on day 11, which was 1.8- and 2.9-times higher than the peak expression level in spheroid homo-culture control in serum-free (day 3) and serum-containing media (day 4), respectively. The albumin secretion function was maintained for at least two weeks; it was 5.1 (in serum-free medium) and 17.8 (in serum-containing medium) times higher than spheroid homo-culture on day 13. Similarly, the co-culture system also expressed approximately 5.5- and 3.1-times higher 3-methylcholanthrene-induced cytochrome P450 enzymatic activity on day 14 as compared to the homo-culture control in serum-free and serum-containing medium, respectively. In conclusion, this unique co-culture system demonstrated the synergistic roles of homotypic cell-cell interaction, heterotypic cell-cell interaction, cell-substrate interaction and soluble stimuli in hepatocyte functional maintenance.
authors.affiliation: Division of Biomedical Sciences, Johns Hopkins in Singapore, Singapore.
authors.forename: Hong-Fang
authors.initials: HF
authors.lastname: Lu
authors.forename: Kian-Ngiap
authors.initials: KN
authors.lastname: Chua
authors.forename: Peng-Chi
authors.initials: PC
authors.lastname: Zhang
authors.forename: Wei-Seng
authors.initials: WS
authors.lastname: Lim
authors.forename: Seeram
authors.initials: S
authors.lastname: Ramakrishna
authors.forename: Kam W
authors.initials: KW
authors.lastname: Leong
authors.forename: Hai-Quan
authors.initials: HQ
authors.lastname: Mao
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D002454:Cell Differentiation; D049109:Cell Proliferation; D048429:Cell Size; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D019164:Liver, Artificial; D008297:Male; D008422:Materials Testing; D051379:Mice; D041681:NIH 3T3 Cells; D011145:Polyvinyls / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D018874:Spheroids, Cellular / Q000166:cytology / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*; D005465:Fluorocarbon Polymers
chemical_list: D001672:Biocompatible Materials; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers