Anticancer drug discovery efforts have used 2‐D cell‐based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3‐D cell culture models are expected to bridge the gap between 2‐D and in vivo models. However, 3‐D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large‐sized spheroids. In addition, cancer cells cultured with FP001‐containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage‐independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3‐D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular‐targeted anticancer drugs. Three‐dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.

Mesenchymal stem cell (MSC) transplantation, in particular allogeneic transplantation, is a promising therapy for a variety of diseases. However, before performing allograft treatment it is necessary to find suitable donors, establish culture methods that maintain cell quality, and reduce cell production costs. Here, we present a new method of producing allogeneic MSCs combining human umbilical cord-derived mesenchymal stem cells (UCMSCs) and chitin-based polysaccharide fibers (Cellhesion^® MS). UCMSC numbers significantly increased, and cells grew as dispersed spheres on Cellhesion^® MS. Subsequent biological analyses showed that the expression levels of stemness-related and migration-related genes were significantly upregulated, including octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and C-X-C chemokine receptor type 4 (CXCR4). The secretion levels of paracrine factors such as prostaglandin E2 (PGE₂), TNFα-stimulating gene (TSG)-6, fibroblast growth factor 2 (bFGF), and Angiogenin (Ang) from UCMSCs using Cellhesion^® MS were significantly higher than with microcarrier and U-bottom plate culture. In addition, culture supernatant from UCMSCs with Cellhesion^® MS had better angiogenic potential than that from monolayer cultured UCMSCs. Furthermore, we succeeded in a scaled-up culture of UCMSCs with Cellhesion^® MS using a closed culture bag. Therefore, Cellhesion^® MS is a key material for producing high-quality UCMSCs in a three-dimensional (3D) culture system.

The evaluation of drug pharmacokinetics in the small intestine is critical for developing orally administered drugs. Caucasian colon adenocarcinoma (Caco-2) cells are employed to evaluate drug absorption in preclinical trials of drug development. However, the pharmacokinetic characteristics of Caco-2 cells are different from those of the normal human small intestine. Besides this, it is almost impossible to obtain primary human intestinal epithelial cells of the same batch. Therefore, human iPS cell-derived enterocytes (hiPSEs) with pharmacokinetic functions similar to human intestinal epithelial cells are expected to be useful for the evaluation of drug absorption. Previous studies have been limited to the use of cytokines and small molecules to generate hiPSEs. Dietary fibers play a critical role in maintaining intestinal physiology. We used gellan gum (GG), a soluble dietary fiber, to optimize hiPSE differentiation. hiPSEs cocultured with GG had significantly higher expression of small intestine- and pharmacokinetics-related genes and proteins. The activities of drug-metabolizing enzymes, such as cytochrome P450 2C19, and peptide transporter 1 were significantly increased in the GG treatment group compared to the control group. At the end point of differentiation, the percentage of senescent cells increased. Therefore, GG could improve the differentiation efficiency of human iPS cells to enterocytes and increase intestinal maturation by extending the life span of hiPSEs.

Scalable production of avian suspension cell exhibits a valuable potential on therapeutic application by producing recombinant protein and as the substrate for virus growth. This study sought to establish a system with chemically defined components for three-dimensional (3D) culture of chicken primordial germ cells (cPGCs), a pluripotent avian cell type. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium, and cPGCs did not sediment,and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1week of culture in 3D-FAot medium, an aseric chemically defined medium containing FP003, indicating that this medium enhances the expansion of cPGCs. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. cPGCs harboring both PGK:EGFP and VASA:tdTOMATO robustly expressed both fluorescent proteins upon culture in 3D-FAot, suggesting that this approach is perspective for recombinant protein production. In summary,this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring or loss of cellular properties. This system provides a platform for large-scale culture ofcPGCs in industry.

Suspension culture is superior to planar culture in efficiently growing large numbers of cells. It also greatly improves cell functions by mimicking the in vivo environment. Although several methods are available to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells, their differentiation by suspension culture by using functional polymers remains unreported. We developed a suspension culture system using the functional polymers FP001 and FP003 to generate hepatocyte-like cells with superior phenotypes than human iPS cell-derived hepatocyte-like cells generated in planar culture. Specifically, optimizing the initial spheroid size and utilizing FP003 effectively maintained appropriate spheroid structures, resulting in higher albumin production compared to FP001. In 3D sphere culture, the mRNA expression of albumin (ALB), drug-metabolizing enzymes and transporters, and the activity of CYP1A2 were increased in the presence of 0.01% FP003. Additionally, immunofluorescence analysis confirmed uniform hepatic differentiation throughout the spheroids. These findings suggest that differentiation in 3D sphere culture with the functional polymer FP003 improved the function of human iPS cell-derived hepatocyte-like cells. Therefore, we conclude that this system may be an effective strategy to generate human iPS cell-derived hepatocyte-like cells with enhanced functions.