Three-Dimensional In Vitro Hepatic Constructs Formed Using Combinatorial Tapered Stencil for Cluster Culture (TASCL) Device

Abstract

Attempts to create artificial liver tissue from various cells have been reported as an alternative method for liver transplantation and pharmaceutical testing. In the construction of artificial liver tissue, the selection of the cell source is the most important factor. However, if an appropriate environment (in vitro/in vivo) cannot be provided for various cells, it is not possible to obtain artificial liver tissue with the desired function. Therefore, we focused on the in vitro environment and produced liver tissues using MEMS technology. In the present study, we report a combinatorial TASCL device to prepare 3D cell constructs in vitro. The TASCL device was fabricated with an overall size of 10 mm × 10 mm with microwells and a top aperture (400 µm × 400 µm, 600 µm × 600 µm, 800 µm × 800 µm) and bottom aperture (40 µm × 40 µm, 80 µm × 80 µm, 160 µm × 160 µm) per microwell. The TASCL device can be easily installed on various culture dishes with tweezers. Using plastic dishes as the bottom surface of the combinatorial TASCL device, 3D hepatocyte constructs of uniform sizes (about ɸ 100 μm-ɸ 200 μm) were produced by increasing the seeding cell density of primary mouse hepatocytes. The 3D hepatocyte constructs obtained using the TASCL device were alive and secreted albumin. On the other hand, partially adhered primary mouse hepatocytes exhibited a cobblestone morphology on the collagen-coated bottom of the individual microwells using the combinatorial TASCL device. By changing the bottom substrate of the TASCL device, the culture environment of the cell constructs was easily changed to a 3D environment. The combinatorial TASCL device described in this report can be used quickly and simply. This device will be useful for preparing hepatocyte constructs for application in drug screening and cell medicine.

Keywords: Biomedical microdevices; Hepatic constructs; Primary hepatocytes; Tapered stencil for cluster culture (TASCL) device; Three-dimensional (3D) culture.

Figure 1

Fabrication of combinatorial TASCL device. (A) The combinatorial TASCL device consisted of various microwells measuring 10 mm by 10 mm with a thickness of 0.55 mm. Scale bar: 10 mm. (B) The combinatorial TASCL device was observed under phase-contrast microscopy. Scale bar: 500 μm. (C) A schematic diagram of cell seeding and cell construct process using TASCL. Cell constructs can be created under multiple initial conditions simultaneously by injecting cell suspension onto the TASCL device.

Figure 2

Phase-contrast photomicrographs of primary mouse hepatocyte constructs created using a combinatorial TASCL device and the bottom surface of the TASCL device employed as a 35-mm uncoated plastic culture dish. Primary mouse hepatocytes at a density of 5 × 10⁴ (A, D, G, and J), 1 × 10⁵ (B, E, H, and K), and 2 × 10⁵ cells (C, F, I, and L) were inoculated on the combinatorial TASCL device for 6 days. The culture periods are as follows: 1 day (A, B, and C), 2 days (D, E, and F), 3 days (G, H, and I), and 6 days (J, K, and L). Scale bar: 200 µm.

Figure 3

Phase-contrast photomicrographs of primary mouse hepatocyte constructs created using combinatorial TASCL device and the bottom surface of the TASCL device employed as a 35-mm collagen-coated culture dish. Primary mouse hepatocytes at a density of 5 × 10⁴ (A, D, G, and J), 1 × 10⁵ (B, E, H, and K), and 2 × 10⁵ cells (C, F, I, and L) were inoculated on a combinatorial TASCL device for 6 days. The culture periods were as follows: 1 day (A, B, and C), 2 days (D, E, and F), 3 days (G, H, and I), and 6 days (J, K, and L). Scale bar: 200 µm.

Figure 4

Morphological examination of primary mouse hepatocyte created using the TASCL device or a collagen-coated culture dish. Hepatocytes were cultured on the TASCL device (hepatocyte constructs) and a collagen-coated culture dish (2D monolayer culture). A total of 2 × 10⁵ cells were seeded on the TASCL device and cultured for 2 days. A 35-mm plastic culture dish was used as the bottom part of the TASCL device. The morphology of the hepatocytes treated with 2D monolayer culture is shown after 48 h of culture (A, B, and C). The morphology of the hepatocyte constructs is shown (D, E, and F). (C, G, H, and I) Fluorescent photomicrographs of (B, D, E, and F) after staining with calcein AM. Scale bars: 500 μm (A, D, and G), 200 μm (B, C, E, and H), and 100 μm (F and I).

Figure 5

Albumin secretion in primary mouse hepatocyte constructs created using the TASCL device. The level of albumin synthesis was measured after 24 h of accumulation. (A) Hepatocytes were incubated on the TASCL device for 24 h. (B) Hepatocytes were incubated on the collagen-coated dishes for 24 h. The data represent the mean and SD of three independent experiments.