Integrating Oxygen and 3D Cell Culture System: A Simple Tool to Elucidate the Cell Fate Decision of hiPSCs.

Oxygen, as an external environmental factor, plays a role in the early differentiation of human stem cells, such as induced pluripotent stem cells (hiPSCs). However, the effect of oxygen concentration on the early-stage differentiation of hiPSC is not fully understood, especially in 3D aggregate cultures. In this study, we cultivated the 3D aggregation of hiPSCs on oxygen-permeable microwells under different oxygen concentrations ranging from 2.5 to 20% and found that the aggregates became larger, corresponding to the increase in oxygen level. In a low oxygen environment, the glycolytic pathway was more profound, and the differentiation markers of the three germ layers were upregulated, suggesting that the oxygen concentration can function as a regulator of differentiation during the early stage of development. In conclusion, culturing stem cells on oxygen-permeable microwells may serve as a platform to investigate the effect of oxygen concentration on diverse cell fate decisions during development.

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.

Efficient High-Density hiPSCs Expansion in Simple Dialysis Device.

iPSCs are potential cell types that can be used for regenerative medicine. Suspension culture is the main approach to produce a sufficient amount of required cells to realize the application of hiPSCs for the transplantation of the large organ. The dialysis culture system holds the potential to reduce the cost by utilizing endogenous growth factors and recycle the remaining exogenous growth factors at the same time. However, the current large scale dialysis culture system was not optimized for expanding the high-density culture. Several problems such as the requirement of the large volume and technical complexity make the optimization of this system remain challenging. Also, the interference of mechanical stress in the dynamic suspension culture may reduce cellular viability, pluripotency, and differentiation capacity. Here, we describe the simple miniaturized dialysis platform to evaluate the feasibility of high-density hiPSCs culture by combining the utilization of endogenous growth factors supported by a continuous exchange of nutrition and toxic metabolic product in a low mechanical stress culture environment in a viscoelastic medium.

Differentiation of Human Induced Pluripotent Stem Cells into Definitive Endoderm Using Simple Dialysis Culture Device.

Therapeutic use of differentiated organ cells from human induced pluripotent stem cells (hiPSCs) is one of the promising strategies for regenerative medicine. Differentiation into definitive endoderm is an essential process in the preparation of metabolic organs. However, the manufacturability of differentiation is limited due to the high-cost cytokines required for the differentiation of endodermal lineage. Furthermore, the cytokines remaining in the used culture medium and possible endogenous factors are removed along with toxic metabolites by the medium replacement. To address these problems, the application of dialysis culture can retain and fully utilize their accumulation to create a better culture environment that contributes to differentiation cost reduction.

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.

Production of homogenous size-controlled human induced pluripotent stem cell aggregates using ring-shaped culture vessel.

Aggregate size is an important parameter that determines the cell fate and quality of the resulting human-induced pluripotent stem cells (hiPSCs). Nowadays, large-scale suspension culture is a common method for scaling-up the biomanufacturing of hiPSCs to realize their practical application. However, this culture system exhibits a complex hydrodynamic condition resulting from the different mixing conditions of culture media, which potentially produce non-uniform aggregates, which may decrease the quality of the cell yield. Here, we performed expansion in a ring-shaped culture vessel and compared it with three other suspension-based culture systems to evaluate the uniformity and characteristics of hiPSC aggregates. Morphologically, the hiPSC aggregates formed and expanded in the ring-shaped culture vessel, resulting in small and uniform aggregates compared to the other culture systems. This aggregate population showed a decent mass transfer required for the exchange of biochemical substances, such as nutrients, growth factors, oxygen, and waste metabolic products, inside the aggregates. Thus, better metabolic performance and pluripotency markers were achieved in this system. Interestingly, all culture systems used in this study showed different tendencies in embryoid body differentiation. The smaller aggregates produced by sphere ring and dish bag tended to differentiate toward ectodermal and mesodermal lineages, while predominantly larger aggregates from the 6-well plates and spinner flask exhibited more potential for endodermal lineage. Our study demonstrates the production of a decent homogenous aggregate population by providing equal hydrodynamic force through the ring-shaped culture vessel design, which may be further upscaled to produce a large number of hiPSCs for clinical applications.

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.

Protection of human induced pluripotent stem cells against shear stress in suspension culture by Bingham plastic fluid.

Suspension culture is an important method used in the industrial preparation of pluripotent stem cells (PSCs), for regenerative therapy and drug screening. Generally, a suspension culture requires agitation to keep PSC aggregates suspended and to promote mass transfer, but agitation also causes cell damage. In this study, we investigated the use of a Bingham plastic fluid, supplemented with a polysaccharide-based polymer, to preserve PSCs from cell damage in suspension culture. Rheometric analysis showed that the culture medium gained yield stress and became a Bingham plastic fluid, after supplementation with the polymer FP003. A growth/death analysis revealed that 2 days of aggregate formation and 2 days of suspension in the Bingham plastic medium improved cell growth and prevented cell death. After the initial aggregation step, whereas strong agitation (120 rpm) of a conventional culture medium resulted in massive cell death, in the Bingham plastic fluid we obtained the same growth as the normal culture with optimal agitation (90 rpm). This indicates that Bingham plastic fluid protected cells from shear stress in suspension culture and could be used to enhance their robustness when developing a large-scale.

Size-dependent hepatic differentiation of human induced pluripotent stem cells spheroid in suspension culture.

Suspension culture of three-dimensional (3D) spheroid of human induced pluripotent stem cells (hiPSCs) has been known as a potential method to enhance the scalability of hepatic differentiation of hiPSCs. However, the impact of size-related factor of initial formed spheroid were not largely considered. To address this problem, we evaluate the impact of different specific spheroid size of hiPSCs by forming the individual spheroid from different number of hiPSCs and differentiated into hiPSCs-derived hepatocytes (iHeps). The results showed that larger spheroid exhibit enhanced capability to differentiated into hepatic lineage by increasing the expression marker albumin, CYP3A4 and lower expression of fetal hepatic marker AFP. Several factor such as the tendency of cystic like structure forming, the necrotic area of the large dense spheroid, and interference of WNT/ß-catenin signaling was significantly affecting the resulted iHeps. In this study, we suggest that the optimal spheroid size for hepatic differentiation can be attained from 500 to 600 µm diameter spheroid formed from 12,500-25,000 hiPSCs. This size can be potentially applied for various practical use of hepatic differentiation in scalable suspension culture.