Construction of Artificial Hepatic Lobule-Like Spheroids on a Three-Dimensional Culture Device

Abstract

One major purpose of cell culture is the reconstruction of physiological structures. Using bovine aortic epithelium cell line HH (JCRB0099) as feeder cells and rat primary hepatocytes, we constructed hepatic lobule-like spheroids on a cell array plate designed for three-dimensional (3D) culture. Microfabricated patterning of the cell array with poly(ethyleneglycol) brushes promotes the formation of spheroids at 100-μm diameter at 100-μm intervals. Our standard protocol is to seed with feeder HH cells and then seed with primary hepatic parenchymal cells. The composite cell spheroids thus obtained are called heterospheroids. Feeder cells that were attached to the plate migrated and encompassed the spheroidal hepatocyte mass. Electron microscopy revealed Disse space-like structures characterized by hepatocyte-rooted microvilli rooted between hepatocyte and feeder epithelial HH cells. Differentiated hepatic functions such as albumin synthesis and cytochrome P450 subfamily CYP3A activities were maintained for 28 days in the heterospheroid versus monospheroid and monolayer cultures. In addition, glucuronide conjugation activity was maintained at a high level in heterospheroids. These results indicate that structurally similar hepatic lobules were formed in a microfabricated cell array coculture system and that the culture conditions are beneficial for maintaining differentiated hepatic functions.

Keywords: Artificial hepatic lobules; Hepatic function; Three-dimensional culture.

Figure 1

Microscopic appearance of the cell culture surface of a cell array (A, B) and schematic illustration of 3D coculture spheroid (C). Original magnifications: 25× (A) and 50× (B). Scale bars: 100 μm (A, B). (A) An arrow indicates the edge of the well. (B) The magnification of the square in (A). The cell culture area is inside the clear circle coated with collagen type I, and the outer moiré-like pattern is the surface covered with superhydrophilic poly(ethyleneglycol) molecules that inhibit the attachment of proteins and cells. (C) Yellow and light pink cells represent primary hepatic parenchymal cells and bovine aortic HH cells (feeder). P, poly(ethyleneglycol) layer.

Figure 2

Hepatocyte–feeder cell heterospheroids formed on the cell array. Rat hepatocytes were cultured for 48 h on the feeder cell–precultured cell array. Phase contrast micrograph of a downward view (A) and horizontal section stained with toluidine blue (B, C). Uniform spheroids were cultured regularly on the cell array. A typical spheroid is shown in (C). Arrowheads indicate HH cells that migrated from the culture plate and enwrapped spheroidal hepatocyte masses. Scale bars: 100 μm (A, B) and 10 μm (C).

Figure 3

Electron micrograph of hepatocyte (Hp) and feeder (HH) cell contact area. Disse space-like structure with hepatocyte-rooted microvilli was observed (A). m, mitochondria. (B) Magnification of square in (A). Arrows in (B) indicate the microvilli.

Figure 4

Time course changes in albumin synthesis (A) and drug-metabolizing activities (B) of rat primary hepatocytes cultured under various conditions. Hepatocytes were cultured on a cell array with feeder cells (heterospheroid), without feeder cells (monospheroid), and on a conventional culture plate (monolayer). Albumin synthesis was measured by 24-h accumulation. Testosterone 6-β-hydroxylation was determined by 4-h incubation with 100 μmol/L testosterone. Each point indicates the mean ± SD (n = 3).