Three-dimensional (3D) tumor models have been established for more precise drug discovery in vitro. We previously demonstrated an in vitro 3D tumor model using silicate fiber scaffold (SFs) to assess the efficacy of potential anticancer drugs. However, mechanical forces, such as shear stress, which are known to regulate cell behavior in tissues, were absent in the model. In the present study, we developed a novel microfluidic chip that is based on a computational flow simulation. The microfluidic chip was prepared with a 3D computer aided design (CAD)/printer. The human colon adenocarcinoma cell line (HT-29) on SFs formed a mature 3D structure in this perfusion culture system and showed time-dependent drug uptake. In addition, this culture system allowed for control of the intensity of shear stress to the 3D cells by changing the fluid flow. The expression of tumor development-related genes, such as ATP synthase and CXCS4, was induced by fluid flow shear forces. This technology provides a valuable tool for future drug screening and metastasis studies.

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Improvement of three-dimensional (3D) culturing conditions, including the substrates used for cell growth, is needed for a variety of cell-based applications as alternatives to animal testing and experimentation. In this study, we synthesized a nonwoven fabric comprised of polyacrylonitrile nanofibers of several widths for use as scaffolds in 3D culturing, and evaluated the optimal nanofiber width to achieve growth and function of human hepatoblasts derived from human fetal hepatocytes during static culturing. For these analyses, several nanofibers with widths between 170–1300 nm were generated by electrospinning. When cultured within 600 nm nanofiber scaffolds, hepatoblasts derived from human fetal hepatocytes exhibited a spherical shape. Furthermore, these cells exhibited enhanced cellular CYP3A4 activity when cultured in 300 nm and 600 nm nanofibers, reaching 2.20 ± 0.05 pmol/10⁶ cells/min, and 2.63 ± 0.06 pmol/10⁶ cells/min, respectively, which were comparable to those of primary human hepatocytes harvested from livers of adult donors. Conversely, hepatoblasts exhibited a flat shape and relatively low CYP3A4 activity when cultured within 170 nm, 1000 nm, and 1300 nm nanofibers. These results suggest that there is a link between the shape and activity of hepatocytes, and that our nonwoven fabric made with polyacrylonitrile nanofibers comprises a promising scaffold for 3D culturing of human hepatocytes.

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