Development of a 3D Cell Culture System Using Amphiphilic Polydepsipeptides and Its Application to Hepatic Differentiation

Abstract

Various three-dimensional (3D) culture systems are available to provide more accurate in vivo mimicry than two-dimensional (2D) cultures. Synthetic and/or xeno-free biomaterials are desired, as they would provide lower batch-to-batch variability and high repeatability. Here, we introduce a 3D culture system using nanofibers composed of an amphiphilic polydepsipeptide-based polymer named HYDROX, which turns into 3D nanofibers after hydration. Our system produces a large amount of cell aggregates and requires only the seeding of a cell mixture. In addition, cells cultured with HYDROX can be collected with only a centrifugation procedure, and analytical assays can then be performed. Here, we applied HYDROX to hepatic differentiation from induced pluripotent stem cells. The cells cultured with HYDROX formed aggregates and HYDROX strongly promoted hepatic differentiation and maturation in terms of functions such as the positive ratio of alpha-1 antitrypsin, the production of albumin, the cytochrome P450 (CYP) 3A4 activity, and the low-density-lipoprotein uptake ability. In addition, primary human hepatocytes cultured with HYDROX showed significantly improved CYP3A4 gene expression and activity. The viscoelasticity and stiffness of HYDROX can be modulated by varying the concentration of the synthetic polymer, thereby providing a suitable microenvironment for the differentiation of cells with various characteristics toward a target cell type. Our findings demonstrated that HYDROX is a promising biomaterial for 3D cultures in research fields ranging from basic cell research to drug discovery.

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research

“…Our 3D culture system using HYDROX has fairly high usability, since only a simple process of adding the cell suspension to the dried gel-coated plate is needed to obtain cell aggregates. Org-HYDROX can be separated from the HYDROX with only centrifugation, allowing the subsequent assays to be performed easily and conveniently 21 . Therefore, due to its high usability and high hepatic functions that could be maintained up to 35 days, Org-HYDROX could be a promising in vitro model in the field of pharmaceutical research.…”

“…We developed a 3D nanofiber biomaterial based on this polymer, named HYDROX, and showed that it was applicable to the 3D culture of various cell types. In addition, we demonstrated that the HYDROX-culture system could be used to promote the differentiation and maturation of human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells 21 . Given these advantages, we hypothesized that HYDROX could be a promising xeno-free biomaterial for human liver orgnaoids culture and differentiation toward hepatocytes.…”

Current Advances in 3D Dynamic Cell Culture Systems

“…In another study, a 3D culture system using amphiphilic polydepsipeptides (HYDROX) was found to promote the differentiation of induced pluripotent stem cells (iPSCs) into the hepatic cell. Meanwhile, CYP3A4 , an important metabolic enzyme-gene, which mostly expressed in human liver, was upregulated in primary human hepatocytes cultured with HYDROX, and these cells showed higher activity compared with those cultured in the 2D system [ 37 ]. Cells culture methods also determined the expression of genes associated with cytoskeletal protein, contractility, and matrix remodelling [ 24 ].…”