Hepatocyte spheroid arrays inside microwells connected with microchannels

Abstract

Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications.

Figure 1

Schematics of the fabrication of a SM chip with microchannels. (a) Preparation of a stamp for microcontact printing. Concave wells of 100 μm head diameter were constructed in a PMMA plate, with which a PDMS stamp was molded. (b) Modification of microwells for cell culture. Microwells 300 μm in diameter and 400 μm in height were connected to channels 100 μm wide and 100 μm deep. The entire surface of the chip, including microwells, was coated with a thin platinum layer. The PDMS stamp was inked with 1 mM RGD peptide and microscopically contacted with the center of the bottom faces of the microwells to create cell adhesion regions. The chip was immersed in 5 mM PEG-SH in ethanol solution to obtain a cell nonadhesion region around the RGD peptide stamped regions. (c) Spheroid formation in a microwell.

Figure 2

SM chips. (a) Batch-type SM chip. (b) Flow-type SM chip. (c) PDMS stamp for microcontact printing. (d) Pattern configuration of cell adhesion regions constructed by microcontact printing in the batch-type SM chip, demonstrated using a red fluorescent paint. (e) Two-dimensional CAD drawing of the flow-type SM chip. (f) Microwells connected to microchannels in the flow-type SM chip, visualized with red and green fluorescent solutions.

Figure 3

Spheroid formation in the SM chips. (a) Primary hepatocytes seeded in the batch-type SM chip. (b) Hepatocytes spontaneously formed spheroids with a uniform diameter at the center of each microwell during 2 days of rotational culture. Hepatocyte spheroids in the batch-type (c) and the flow-type (d) SM chips during 7 days of rotational culture. Hematoxylin and eosin staining of vertical cross sections (e) and Masson trichrome staining of a horizontal cross section (f) of representative spheroids in the batch-type SM chip during 3 days of rotational culture.

Figure 4

Dependence of EROD metabolism of hepatocyte spheroids on the concentration of 3-methylcholanthrene in a batch-type SM chip during 10 days of rotational culture. The values and error bars represent the mean and standard deviation, respectively, of three independent experiments.

Figure 5

Four AROD activities measured simultaneously with the flow-type SM chip. (a) Representative fluorescence microscopy image. The red fluorescence of resorufin, which was obtained by the conversion of ethoxy-, methoxy-, pentoxy-, and benzyloxy-resorufin, was detected on a single chip. (b) Comparison of four AROD activities. The values and error bars represent the mean and standard deviation, respectively, of three independent experiments.