Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device

Abstract

In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 µm × 500 µm) and a bottom aperture (300 µm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.

Keywords: Human hepatocellular carcinoma (HepG2) cells; Medical evaluation; Spheroid; Tapered stencil for cluster culture (TASCL); Three-dimensional (3D) culture.

Figure 1

Fabrication of the Tapered Stencil for Cluster Culture (TASCL) device. (A) The TASCL device consisted of microwells measuring 10 mm × 10 mm with a thickness of 0.55 mm. Each microwell in the TASCL device had a top aperture (500 µm × 500 µm) and bottom aperture (300 µm diameter circular). The TASCL device was observed under phase-contrast microscopy. Scale bars: 1 mm and 200 µm. (B) A schematic diagram of cell seeding and the cluster formation process using the TASCL device. Human hepatocellular carcinoma cell (HepG2) spheroids can be created under multiple initial conditions simultaneously by injecting the cell suspension onto the TASCL device. (C) The study protocol for spheroid formation of HepG2 cells using the TASCL device. DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; SEM, scanning electron microscope.

Figure 2

Phase-contrast photomicrographs of the HepG2 spheroids created using the TASCL device. HepG2 cells at a density of 4 × 10⁴ (A, D, and G), 2 × 10⁵ (B, E, and H), and 4 × 10⁵ cells (C, F, and I) were seeded onto the TASCL device (calculated to be 100, 500 and 1,000 cells/microwell each) and then cultured for 4 days. The culture periods were as follows: day 1 (A, B, and C), day 2 (D, E, and F), and day 4 (G, H, and I). Scale bar: 500 µm.

Figure 3

Efficiency of the formation, recovery, and size of HepG2 spheroids in the TASCL device. HepG2 cells at density of 4 × 10⁴, 2 × 10⁵, and 4 × 10⁵ cells/TASCL were inoculated on the TASCL device for 2 days. (A) The efficiency of spheroid formation was evaluated. (B) The efficiency of spheroid recovery was evaluated. (C) The size of the formed spheroids was measured in the case of 100, 500, and 1,000 cells/droplet. Data are shown as the mean ± standard deviation for triplicate experiments.

Figure 4

Scanning electron microscope (SEM) images of the HepG2 spheroids in the TASCL device. HepG2 cells at a density of 4 × 10⁵ were inoculated on the TASCL device for 2 days. (A, B) Phase-contrast photographs of the HepG2 spheroids observed from the microwell in a TASCL device. (C) SEM image observed of the microwell in a TASCL device. (D) SEM image of HepG2 spheroids (artificial red color) observed within the microwell of a TASCL device. The spheroids in the microwells exhibited perfectly spherical aggregation with a diameter and size of 218 ± 10.1 µm and 5.4 ± 0.69 × 10⁶ µm³, respectively. Data are shown as the mean ± standard deviation for triplicate experiments.

Figure 5

The live/dead cells and single cell viability in the HepG2 spheroids created using the TASCL device. (A) HepG2 cells at a density of 4 × 10⁴, 2 × 10⁵, and 4 × 10⁵ cells were inoculated on the TASCL device for 2 days (phase contrast; a, b, and c). HepG2 spheroids were stained with calcein AM and EthD-1 to visualize live and dead cells. Calcein acetoxymethyl ester (calcein AM)-positive green fluorescence (d, e, and f) in live cells and ethidium homodimer-1 (EthD-1)-positive red fluorescence (g, h, and i) in dead cells were observed with a fluorescence microscope. (B) After trypsin treatment, single cell survival was measured using a trypan blue exclusion assay. Data are shown as the mean ± standard deviation of triplicate values. *p < 0.05.

Figure 6

Albumin secretion in the HepG2 spheroids created using the TASCL device. HepG2 cells at density of 4 × 10⁴, 2 × 10⁵, and 4 × 10⁵ cells/TASCL were seeded onto the TASCL device and then cultured for 4 days. The amount of albumin secretion in the medium was measured by ELISA after 24 h of accumulation on day 2 (A) and day 4 (B) of culture. Data are shown as the mean ± standard deviation for triplicate experiments. *p < 0.01.