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

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

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

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

Designing a banking scale of human induced pluripotent stem cells based on suspension time-dependent quality variations in filling and cryopreservation processes

To establish a robust commercial production system for a cell product, it is necessary to investigate a lot of variable factors inside and outside of the system and discuss the cell manufacturability. In case of trying a scale-up of banking system for human induced pluripotent stem cells (hiPSCs), the process time to fill the cell suspension into vials before cryopreservation is prolonged. And that will cause the decay of the cell quality, because cryoprotective agent (CPA) including dimethyl sulfoxide has toxicity to cells. Based on such fluctuation of cell product quality derived from time-dependency in down-stream process, novel strategy to design a process time and a banking scale is required. In this study, four performance indexes, survival ratio of cells during suspension in CPA before cryopreservation (γ), survival ratio, attachment efficiency and specific growth rate of cells after cryopreservation (β, α and μ, respectively) are proposed to evaluate the cellular state and potential of the product. And, the quality variations of suspended cells in CPA are elucidated by changing the process time of suspension at room temperature and 4 °C. At room temperature, γ decreased with process time (ts) exponentially, being γ = 0.72 at ts = 6 h. With respect to α, 4 hours suspension at room temperature had an insignificant effect, however, it dropped after the lag-time, being α = 0.73 at ts = 6 h. In contrast, β and μ were kept high level of 0.80 and 5.3 × 10-2 h-1, respectively, similarly to those without the process. In addition, the suspension at 4°C made the enhancement of γ and α at ts = 6 h (γ = 0.88 and α = 1.08, respectively), suggesting that the suppression in cell activity during suspension is important to preserve the cell quality. In conclusion, the proposed performance indexes are useful to estimate the state and potential of cell product in filling and cryopreservation processes, and the temperature control in filling process is one of the promising factors to maintain the cell product quality. Please click Additional Files below to see the full abstract

Internal Structure and Viscoelasticity Estimation by a Mechanical Impedance - In case of a vibrating disk -

In a stiffness estimation of living body, an internal structure under the skin influences the measured results. Because a different stiffness of body caused by bones and muscles is obtained. In this paper, by using a measurement system of mechanical impedance, the relations between a viscoelasticity and a distance from the surface of silicone-gel model is calculated. This relation is applied to silicone-gel tumor model and a shape and a viscoelasticity of semi-sphere silicone-gel tumous are estimated. The obtained results are expressed as a reconstructed 3-D image of shape / viscoelasticity. The revised curve-fitting of mechanical impedance and the cancellation of peripheral vibration influence are proposed in order to increase an estimation acccuracy

Tooth Mobility Measurement of Dental Implants

The use of dental implants has increased together with increases in the human life span and it has become an imperative subject for dentists to familiarize themselves with this treatment modality. Unfortunately, there has been no practical and quantitative method for in vivo evaluation of the stability of dental implants. In the tooth mobility examination, the tactile sense of natural teeth is different from that of dental implants. The authors have developed an automatic diagnosis system of tooth mobility for clinical use. The biomechanical mobility of peri-implantium is measured with a pseudo-random vibration, from which the viscoelasticity c1, c2, k of peri-implantium is obtained. The diagnosis system has been applied to the quantitative evaluation of the stability of implants : endodontics endosseous implants (titanium pin), endosseous implants (Bioceram). It has also been applied to the evaluation of the long-term prognoses of dental implantation (Bioceram) and the examination of Intramobile implant (IMZ), and the satisfactory results have been obtained

Evidence for the possible involvement of the P2Y6 receptor in Ca2+ mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca2+]i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca2+]i rise via cytoplasmic Ca2+ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 µM) transiently increased [Ca2+]i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y1 receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y6 receptor agonist UDP (200 µM) transiently increased [Ca2+]i and reduced insulin secretion at high glucose, whereas the P2Y2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca2+]i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 µM), U73122 (2 µM, PLC inhibitor), and 2-APB (10 or 30 µM, IP3 receptor antagonist), but neither by staurosporine (1 µM, PKC inhibitor) nor depletion of extracellular Ca2+. The effect of 2-MeSADP on [Ca2+]i was also significantly inhibited by MRS2500, a P2Y1 receptor antagonist. These results suggested that P2Y1 and P2Y6 receptor subtypes are involved in Ca2+ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca2+]i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y1 and P2Y6, but not P2Y2 and P2Y4 receptors