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.

Accurate Evaluation of Hepatocyte Metabolisms on a Noble Oxygen-Permeable Material With Low Sorption Characteristics.

In the pharmaceutical industry, primary cultured hepatocytes is a standard tool used to assess hepatic metabolisms and toxicity in vitro. Drawbacks, however, include their functional deterioration upon isolation, mostly due to the lack of a physiological environment. Polydimethylsiloxane (PDMS) has been reported to improve the function of isolated hepatocytes by its high oxygen permeability when used as a material of microphysiological systems (MPS). However, its high chemical sorption property has impeded its practical use in drug development. In this study, we evaluated a new culture material, 4-polymethyl-1-pentene polymer (PMP), in comparison with PDMS and conventional tissue culture polystyrene (TCPS). First, we confirmed the high oxygen permeability and low sorption of PMP, and these properties were comparable with PDMS and TCPS, respectively. Moreover, using primary rat hepatocytes, we demonstrated maintained high levels of liver function at least for 1 week on PMP, with its low chemical sorption and high oxygen permeability being key factors in both revealing the potential of primary cultured hepatocytes and in performing an accurate evaluation of hepatic metabolisms. Taken together, we conclude that PMP is a superior alternative to both PDMS and TCPS, and a promising material for a variety of drug testing systems.

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.

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.