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.

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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.

Extracellular environments significantly affect cell proliferation, differentiation, and functions. The extracellular environment changes during many physiological and pathological processes such as embryo development, wound healing, and tumor growth. To mimic these changes, we developed novel thiol−maleimide clickable alginate microcapsules, which can introduce thiol-containing peptides by "in situ conjugation" with maleimide-modified alginate, even in serum-containing cell culture media. Additive peptides were rapidly concentrated into microcapsules by a diffusionreaction process in the capsule. The proliferation of encapsulated fibroblasts was accelerated by in situ conjugation of CRGDS, while free RGDS showed no effect. Moreover, encapsulated preosteoblastic cells started osteogenic differentiation via in situ conjugation of BMP-2 mimetic peptides such as CDWIVA and CG-BMP-2 knuckle epitope peptide, while BMP-2 did not induce differentiation of the encapsulated cells. Especially in tissue engineering, accurate and inexpensive methods for inducing cell differentiation are required. We believe that this in situ conjugation approach employing various functional peptides will be useful in biomedical, bioindustrial, and biochemical fields in the future.

Induced pluripotent stem cells (iPSCs) are expected to be an ideal cell source for biomedical applications, but such applications usually require a large number of cells. Suspension culture of iPSC aggregates can offer high cell yields but sometimes results in excess aggregation or cell death by shear stress. Hydrogel-based microencapsulation can solve such problems observed in Suspension culture, but there is no systematic evaluation of the possible capsule formulations. In addition, their biological effects on entrapped cells are still poorly studied so far. We, therefore, immobilized mouse iPSCs in three different types of calcium-alginate (Alg-Ca) hydrogel-based microcapsules; (i) Alg-Ca capsules without further treatment (Naked), (ii) Alg-Ca capsules with poly-l-lysine (PLL) coating (Coated), and (iii) Alg-PLL membrane capsules with liquid cores (Hollow). After 10 days of culture within the medium containing serum and leukemia inhibitory factor, we obtained good cellular expansions (10-13-fold) in Coated and Hollow capsules that were similar to Suspension culture. However, 32 ± 9% of cellular leakage and lower cell yield (about threefold) were observed in Naked capsules. This was not observed in Coated and Hollow capsules. In addition, immunostaining and quantitative RT-PCR showed that the formation of primitive endodermal layers was suppressed in Coated capsules contrary to all other formulations. This agenesis of primitive endoderm layers in Coated capsules is likely to be the main cause of the significantly better pluripotency maintenance in hydrogel-based encapsulation culture. These results are helpful in further optimizing hydrogel-based iPSC culture, which can maintain better local cellular environments and be compatible with mass culture.