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

摘要

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.