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/β-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.

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-β1 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.

Aggregate size is an important parameter that determines the cell fate and quality of the resulting human-induced pluripotent stem cells (hiPSCs). Nowadays, largescale 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 nonuniform 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.

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.

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.