Inflammatory liver tissue formation using oxygen permeable membrane based culture platform.

During chronic liver injury, inflammation leads to liver fibrosis, particularly due to the activation of hepatic stellate cells (HSCs). The involvement of inflammatory cytokines in HSC activation and the interplay among different liver cells are elaborated. To examine their interactions in vitro, many cultured liver tissue models are performed in organoid or spheroid culture with random 3D structure. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-ß), on HSC activation. Gene expression of collagen type I and alpha-smooth muscle actin were enhanced in the hierarchical coculture under lower oxygen tension and TGF-ß1 stimulation. Therefore, this hierarchical in vitro cocultured liver tissue could provide a useful platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.

Dialysis based-culture medium conditioning improved the generation of human induced pluripotent stem cell derived-liver organoid in a high cell density.

Human pluripotent stem cell-derived liver organoids (HLOs) have recently become a promising alternative for liver regenerative therapy. To realize this application, a large amount of human-induced pluripotent stem cells (hiPSCs) derived-liver cells are required for partial liver replacement during transplantation. This method requires stepwise induction using costly growth factors to direct the hiPSCs into the hepatic lineage. Therefore, we developed a simple dialysis-based medium conditioning that fully utilized growth factors accumulation to improve hepatic differentiation of hiPSCs at a high cell density. The results demonstrated that the dialysis culture system could accumulate the four essential growth factors required in each differentiation stage: activin A, bone morphogenetic protein 4 (BMP4), hepatocyte growth factor (HGF), and oncostatin M (OSM). As a result, this low lactate culture environment allowed high-density bipotential hepatic differentiation of up to 4.5 × 107 cells/mL of human liver organoids (HLOs), consisting of hiPSC derived-hepatocyte like cells (HLCs) and cholangiocyte like-cells (CLCs). The differentiated HLOs presented a better or comparable hepatic marker and hepatobiliary physiology to the one that differentiated in suspension culture with routine daily medium replacement at a lower cell density. This simple miniaturized dialysis culture system demonstrated the feasibility of cost-effective high-density hepatic differentiation with minimum growth factor usage.

A miniature dialysis-culture device allows high-density human-induced pluripotent stem cells expansion from growth factor accumulation.

Three-dimensional aggregate-suspension culture is a potential biomanufacturing method to produce a large number of human induced pluripotent stem cells (hiPSCs); however, the use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency, respectively. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device improved cell expansion up to approximately ~3.2 to 4×107 cells/mL. The high-density expansion was supported by reduction of excessive shear stress and agglomeration mediated by the addition of the functional polymer FP003. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous Nodal, which acts as a substitute for depleted transforming growth factor-ß1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, the cost reduced in inverse proportional manner with the cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility of cost-effective mass production of hiPSCs in high-density culture.