Floating or adherent hepatocyte spheroid cultures using microwell chips with polyethylene glycol or polyimide surfaces

Microwell chip culture is a promising technique for controlling spheroid size and producing a large number of homogeneous spheroids. In this study, we focused on the relationship between chip material and the properties of hepatocyte spheroids. The basic chip design was 397 circular microwells, each 400 µm in diameter. Two types of microwell chips were fabricated, coating the bottom surface either with polyethylene glycol (PEG chip) or polyimide (PI chip). Hepatocytes gradually aggregated and formed floating spheroids within each microwell in the PEG chip but formed adherent spheroids within each microwell of the PI chip. Such floating and adherent spheroid morphologies were maintained for at least one month of culture. An explanation for the spheroid formation mechanism is that the plasminogen activator (PA) /plasmin and matrix degradation/remodeling systems were activated in the formation of adherent spheroids. Furthermore, in adherent spheroid cultures, the formation of cell-matrix junctions was promoted, in addition to the development of intercellular junctions. The albumin secretion and drug metabolism activities of the hepatocyte spheroids were higher than those of traditional monolayer hepatocytes, and the adherent spheroids in the PI chip maintained a higher functional expression than the floating spheroids in the PEG chip. Further to this, functional properties of hepatocytes, the expressions of key metabolic enzymes, glucose 6-phosphatase (sugar metabolism), tryptophan 2, 3-dioxygenase (amino acid metabolism), arginase 1 (urea cycle), cytochrome P450 7a1 (lipid metabolism), and cytochrome P450 families (drug metabolism) were evaluated by gene expression analysis. The expression of these key enzymes in hepatocytes was higher in spheroid culture than in general monolayer culture, and the functions of adherent spheroids were superior to those of floating spheroids. These results indicate that the material properties of the microwell chips are important factors that regulate the morphological and functional characteristics of hepatocyte spheroids.