Automated manufacturing for iPSC-derived retinal pigment epithelial cells

Cell manufacturing, which is the most critical steps to realize the transplant of cell-based products for cell therapy or regenerative medicine, will be done in terms of safety, stably and cost-saving under the aseptic environment in the cell processing facility (CPF). The cell processing is regarded as the system consisting of target process, input and output, and there are several fluctuations derived from extrinsic noises (environmental errors) against the system, input quality such as starter cells and materials (medium, reagents, substrate, vessel etc.), and intrinsic disorders (in-process errors) from the behavior variance in manual operation (Fig.1). Especially, intrinsic disorders cause the difficulty to make consistency and robust process for stable quality because the cells have uncertainty accompanied by time-dependent and time-delay properties. Therefore, environmental, material, and operational standardizations are required to realize consistency of processes. In addition, long manufacturing period and small lot size for cell production make the low productivity, causing the high cost production. Please click Additional Files below to see the full abstract

Cell placement in culture vessel after seeding is CPP in induction culture of retina pigment epithelial cells derived from Ipsc

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Comprehensive cell manufacturing system based on flexible modular platform 85

In cell manufacturing, as it is known that the serial processes influence the quality of the cells, the processes in appropriate cell processing facility (CPF) is expected not only to maintain an aseptic environment but also to lead to stable processing. “Design for manufacturability (DFM)” is known to be the general engineering art of designing products in such a way that they are easy to manufacture. This concept exists in almost all engineering disciplines, but the implementation differs widely depending on the manufacturing technology. DFM for cell production will lead to facilitation of the consistency and robustness for process as well as reduction cost for the cell manufacturing. As shown in Fig. 1, the system consists of input and output for the process. There are several fluctuations derived from extrinsic noises (environmental errors) against the system, input quality such as starter cells and materials (medium, reagents, vessel and pipet etc.), and intrinsic disorders (in-process errors). Especially, intrinsic disorders cause the difficulty to make consistency and robust process for stable quality because the cells have uncertainty accompanied by time-dependent and time-delay properties. Therefore, environmental, material, and operational standardizations are required to realize consistent process. A novel design of manufacturing facility has been proposed based on the isolator technology (Fig.2). Our proposal system is the flexible modular platform (fMP) which realize that the individual aseptic modules can connect and disconnect between modules (or pods) flexibly with keeping the aseptic environment in each module (or pods), leading to the compactness of aseptic processing area and quick change-over for multi-purposes and patients. To effectively implement this fMP technology, an interface that can be aseptically detached and attached from one module to another is required, responding to diversified requirements for cell processing. A common tool utilized in isolator based manufacturing of sterile pharmaceuticals is a transfer pod of rapid transfer ports (RTP). However, its interface limited to a circular configuration, and a more versatile aseptic transfer mechanism is sought for handling the connection between modules (or pods). Therefore, the interface of double door system is developed for the flexible connections between modules with shorten of the decontamination process. Furthermore, the standardization of the configuration suggests that the companies, who have novel modules with advanced technologies, lead to taking part in planning for further development of cell processing easily, compared to that in case of monopoly business by a certain company. Thus, our attempts are concluded to build an advanced culture system employing isolator technology, and the adaptation of the fMP in CPF will lead to easy installation of the new modules for production line addition and/or revision through the clinical phases as well as commercial production, which contributes to the reduction of production costs. Please click Additional Files below to see the full abstract

A critical Mach number for electron injection in collisionless shocks

Electron acceleration in collisionless shocks with arbitrary magnetic field orientations is discussed. It is shown that the injection of thermal electrons into diffusive shock acceleration process is achieved by an electron beam with a loss-cone in velocity space that is reflected back upstream from the shock through shock drift acceleration mechanism. The electron beam is able to excite whistler waves which can scatter the energetic electrons themselves when the Alfven Mach number of the shock is sufficiently high. A critical Mach number for the electron injection is obtained as a function of upstream parameters. The application to supernova remnant shocks is discussed.Comment: 4 pages, 2 figure, accepted for publication in Physical Review Letter

High density culture of human induced pluripotent stem cells through the refinement of medium by dialysis in suspension

Human induced pluripotent stem cells (hiPSCs) hold great promise in the field of regenerative medicine for cell-based therapies, tissue engineering, and drug discovery because of their pluripotency and self-renewal capacity. To implement their potential, bio-process developments for robust expansion of hiPSCs are important since large numbers of hiPSCs are required for cell therapy application. Although suspension culture is superior to obtain large numbers of cells, the cost of culturing hiPSC increases with increasing medium consumption, as the culture medium contains many costly macromolecules including basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF-β1), and insulin. Moreover, hiPSCs secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87% of bFGF and 80% of TGF-β1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 106 cells mL-1 was obtained, with 99.5 ± 0.2% OCT 3/4 and 78.3 ± 1.1% TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in conventional suspension culture. Moreover, to obtain high density culture, size- and time-dependent boundary conditions were also considered for the preferable growth of hiPSC in suspension culture. Thus, the concept for high density culture was proposed by considering the boundary conditions for preferable growth of hiPSC, as well as, medium refinement by dialysis to remove toxic metabolites, recycle autocrine factors, and reduce the use of macromolecules for the reduction of culture cost in suspension

The role of Importin-βs in the maintenance and lineage commitment of mouse embryonic stem cells

AbstractMembers of the Importin-β family recognize nuclear localization signals (NLS) and nuclear export signals (NES). These proteins play important roles in various nucleocytoplasmic transport processes in cells. Here, we examined the expression patterns of 21 identified Importin-β genes in mouse embryonic stem cells (mESCs), mouse embryonic fibroblast (MEF) and mESCs differentiated into neural ectoderm (NE) or mesoendoderm (ME). We observed striking differences in the Importin-β mRNA expression levels within these cell types. We also found that knockdown of selected Importin-β genes led to suppression of Nanog, and altered the balance of Oct4/Sox2 expression ratio, which is important for NE/ME lineage choice. Furthermore, we demonstrated that knockdown of XPO4, RanBP17, RanBP16, or IPO7 differentially affected the lineage selection of differentiating mESCs. More specifically, knockdown of XPO4 selectively stimulated the mESC differentiation towards definitive endoderm, while concomitantly inhibiting NE differentiation. RanBP17 knockdown also promoted endodermal differentiation with no effect on NE differentiation. RanBP16 knockdown caused differentiation into ME, while IPO7 knockdown inhibited NE differentiation, without obvious effects on the other lineages. Collectively, our results suggest that Importin-βs play important roles in cell fate determination processes of mESCs, such as in the maintenance of pluripotency or selection of lineage during differentiation

Investigate the stability of seeding process of mesenchymal stem cell on microcarriers by considering heterogeneity

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Development of mass culture process for human induced pluripotent stem cells in suspension

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Comparison of growth activity of bone marrow-derived mesenchymal stem cells in normoxic and hypoxic culture conditions through continuous passage

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