Particle-tracking-based strategy for the optimization of agitation conditions in a suspension culture of human induced pluripotent stem cells in a shaking vessel.

Suspension cultures are widely used for cell expansion in regenerative medicine and production. Shaking culture is one of the useful suspension culture methods that ensures gentle agitation. There are other shaking methods, including orbital shaking, reciprocating, and rocking; however, optimizing the shaking conditions for each method to meet cell culture requirements is time-consuming. In this study, we used a particle-tracking-based strategy for optimizing the agitation conditions. When the average accelerations of aggregates were calculated, high acceleration occurred periodically, and acceleration of the aggregates in orbital shaking was stable. Furthermore, the number of dead cells correlated with the average time of acceleration. We observed that cell growth was ideally maintained by factors such as optimal acceleration, aggregate formation, and cell death. These results indicate that the image-based analyses of aggregates help optimize the agitation conditions for the shaking suspension culture of induced pluripotent stem cells (iPSCs).

Basic points to consider regarding the preparation of extracellular vesicles and their clinical applications in Japan.

In recent years, extracellular vesicles (EVs) have attracted attention as a new therapeutic tool. In Europe, the United States, and Asia, there is an accelerating trend of moving beyond basic research on clinical trials. However, treatment using EVs is still in the research and development stage, and the general public has insufficient awareness and understanding of the risks involved in ensuring safety and efficacy, the status of laws and regulations, and global research and development trends regarding their use. The Japanese Society for Regenerative Medicine, which has promoted the research and development of regenerative medicine, an innovative medical technology based on the principle of delivering it safely, effectively, and promptly, including the establishment of laws and regulations, would like to express two positions in light of the rapid development of therapies using EVs: 1) concern about treatments that are based solely on the discretion of medical practitioners, and 2) active promotion of treatments based on sound scientific evidence. Because EVs are released from cells, there are many similarities between EVs and processed cells in terms of manufacturing processes and safety hazards. As for efficacy, the mechanism of action of EVs is still unclear, as is the case with specified processed cellsb; in such cases, it is difficult to measure potency, identify efficacy-related quality attributes, and evaluate the comparability of quality before and after a change in the manufacturing process. In other words, the number of quality attributes that can be obtained for EVs is limited because of their complex characteristics, and it is difficult to grasp their quality through specifications and characterization. Therefore, while designing a quality control strategy for EVs, it is important to ensure the quality of the final product (EVs) by controlling the raw materials and manufacturing process. On the contrary, since EVs do not contain living cell components and are not classified into specified processed cells, non-commercial clinical research on treatments using EVs and individual medical treatments with EVs at the discretion of medical practitioners are out of the scope of the Act on the Safety of Regenerative Medicine of Japan. At present, there are no relevant laws or regulations for the use of EVs other than the Medical Practitioners' Act and the Medical Care Act in Japan. Therefore, there is a concern that treatment will be performed without sufficient objective evaluation of the scientific basis for safety and efficacy. Despite these concerns, the development of therapies using EVs is underway worldwide. This could potentially lead to a wide variety of new therapeutic areas if the methods needed to stably secure and mass cultivate cells as raw materials and the technologies needed for the mass production of EVs can be developed, in addition to understanding the risks involved and developing relevant laws and regulations. As part of the Japanese Society for Regenerative Medicine, we will continue to work on the development of these methods and technologies and hope that such a promising field will be promoted with a high level of safety before reaching the public.

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.

Apoptosis-based method for determining lot sizes in the filling of human-induced pluripotent stem cells.

Standardization in process design and operation is needed in the commercial production of human-induced pluripotent stem (hiPS) cells. Lot sizing in the filling of hiPS cells into containers, a part of the preservation process, also needs to be standardized because of the temporal changes in cell quality during the process. Here, we present an apoptosis-based method that can determine lot sizes in the filling of hiPS cells considering temporal changes in cell quality. Two indicators were developed for (i) the cell quality change using reactive oxygen species (ROS) measurement and (ii) the cell survival and probability of filling success, which are parts of the lot-sizing problem. Using computational simulation, a map out of the optimal lot size was produced that minimized the expected production costs at a given cell demand and an acceptable change in cell quality. At a filling temperature of 4°C, the largest possible lot size was calculated as 6 L (corresponding to a filling time of 125 min). The results of a sensitivity analysis recommended cold filling or the addition of an antioxidant. The presented method is effective to determine the lot size considering the change in cell quality during filling. The study uniquely combines the experimental results with mathematical modeling and computational simulation techniques. The map out of the optimal lot size could guide the development of industrial filling processes of hiPS cells.

Organization of liver organoids using Raschig ring-like micro-scaffolds and triple co-culture: Toward modular assembly-based scalable liver tissue engineering.

In order to address the remaining issues of fragile structure and insufficient mass transfer faced in modular assembly-based liver tissue engineering, a Raschig ring-like hollowed micro-scaffold was proposed and fabricated using poly-ε-caprolactone with 60% porosity and 11.4 mm2 effective surface area for cell immobilization. The method of cell inoculation, the types of cells for co-culture and the scalability of the proposed hollowed micro-scaffold in perfusion were all investigated to obtain an optimized organoid made of tissue modules. Extracellular matrix was found necessary to establish a hierarchical co-culture, and the triple co-culture of Human Hepatoma Hep G2 cells, liver sinusoid cell line TMNK-1 cells and fibroblasts (Swiss 3T3 cells) was recognized to be the most efficient to obtain higher cell attachment, proliferation and hepatic function. The equipped intersecting hollow channels provided in the micro-scaffold functioned as flow paths to promote mass transfer to the immobilized cells after the modules have been randomly packed into a bioreactor for perfusion culture, and resulted in enhanced albumin production and high cellular viability. Cell density comparable to those found in vivo were obtained in the perfused construct, which also maintained its rigid structure. Those results suggest that modular tissues made with hollowed micro-scaffold-based organoids hold great potential for scaling up tissue engineered constructs towards implantation.

Switching of Cell Proliferation/Differentiation in Thiol-Maleimide Clickable Microcapsules Triggered by in Situ Conjugation of Biomimetic Peptides.

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 diffusion-reaction 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.

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

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.

A novel tool for suspension culture of human induced pluripotent stem cells: Lysophospholipids as a cell aggregation regulator.

Suspension culture for the increase in human induced pluripotent stem cells (hiPSCs) has been one of the major challenges. Previously, we reported that albumin-associated lipids prevented aggregation of hiPSCs, whereas, lipids responsible for this function were unclear. Here, by using cell aggregation assay, we investigated principal lipids regulated aggregation size of hiPSCs. As a result, lysophosphatidic acid (LPA) and Sphingosine-1-phosphate (S1P), known as lysophospholipids acting as a signaling molecule, were identified. These lipids regulated the aggregation size in a dose-dependent manner. Aggregates formed with these lipids kept the high-expression rates of pluripotent marker genes and had the abilities of proliferation. These studies demonstrated that LPA and S1P were useful for suspension culture for hiPSCs without affecting the growth ability and pluripotency of hiPSCs. This knowledge will lead to the development of a simple and robust method for the mass culture of hiPSCs.

Enhanced Hepatic Differentiation of Human Induced Pluripotent Stem Cells Using Gas-Permeable Membrane.

IMPACT STATEMENT: Although oxygen is a vital nutrient for the hepatocytes in vitro, few reports have focused on its effect during hepatic differentiation of induced pluripotent stem cells (iPSCs). In this report, we performed the hepatic differentiation of human iPSCs (hiPSCs) under different atmospheric oxygen concentrations and oxygen supply fluxes to investigate the effects of oxygen in terms of both the concentration and the supply flux. Results demonstrate that direct oxygenation through a polydimethylsiloxane (PDMS) membrane enhances the maturation and efficient production of hiPSC-derived hepatocyte-like cells (iHeps). Thus, direct oxygenation through a PDMS membrane is a better alternative culture method over conventional tissue culture-treated polystyrene (TCPS) plates for the maturation of hiPSC-derived hepatocytes in vitro.

Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells.

Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.

Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture.

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2- to 10-fold increase) without any influence on pluripotency. In addition, albumin-associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009-1016, 2016.

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 µm because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production.

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.

Development of bioactive hydrogel capsules for the 3D expansion of pluripotent stem cells in bioreactors.

Pluripotent stem cells hold great promise for many pharmaceutical and therapeutic applications. However, the lack of scalable methodologies to expand these cells to clinically relevant numbers is a major roadblock in realizing their full potential. To address this problem, we report here a scalable approach for the expansion of pluripotent stem cells within bioactive hydrogel capsules in stirred bioreactors. To achieve rapid crosslinking of cellular microenvironments with tuneable, cell-instructive functionality, we combined calcium-mediated alginate (CaAlg) complexation with crosslinking of poly(ethylene glycol) (PEG) macromers via a Michael-type addition. The resulting hybrid networks have been shown to have very good handling properties and can be readily decorated with biologically active signals such as integrin ligands or Cadherin-based motifs to influence the fate of mouse induced pluripotent stem (iPS) cells. Air-driven co-axial extrusion was used to reproducibly generate gel microcapsules in high-throughput. Furthermore, the gel capsules can be enveloped in a poly(l-lysine) shell to control swelling or molecular permeability independently of the gel composition. iPS cells entrapped within such capsules expanded with limited commitment to the endodermal lineage. Functionalization of gels with an appropriate density of Arg-Gly-Asp (RGD) ligands further increased the iPS cell expansion rate and reduced the spontaneous differentiation. Therefore, the combination of micro-scale instruction of cell fate by an engineered microenvironment and macro-scale cell manipulation in bioreactors opens up exciting opportunities for stem cell-based applications.