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

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

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it’s presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets

Supplementary Video S5. An example simulation result of expansion culture of myoblasts in the case of non-uniform seeding (a=0.1 in Equation 7) from An <i>in silico</i> prediction tool for the expansion culture of human skeletal muscle myoblasts

Regenerative therapy using autologous skeletal myoblasts requires a large number of cells to be prepared for high-level secretion of cytokines and chemokines to induce good regeneration of damaged regions. However, myoblast expansion culture is hindered by a reduction in growth rate owing to cellular quiescence and differentiation, therefore optimization is required. We have developed a kinetic computational model describing skeletal myoblast proliferation and differentiation, which can be used as a prediction tool for the expansion process. In the model, myoblasts migrate, divide, quiesce and differentiate as observed during <i>in vitro</i> culture. We assumed cell differentiation initiates following cell-cell attachment for a defined time period. The model parameter values were estimated by fitting to several predetermined experimental datasets. Using an additional experimental dataset, we confirmed validity of the developed model. We then executed simulations using the developed model under several culture conditions and quantitatively predicted that non-uniform cell seeding had adverse effects on the expansion culture, mainly by reducing the existing ratio of proliferative cells. The proposed model is expected to be useful for predicting myoblast behaviours and in designing efficient expansion culture conditions for these cells

Supplementary Video S1. An example simulation result of expansion culture of myoblasts on the plain surface from An <i>in silico</i> prediction tool for the expansion culture of human skeletal muscle myoblasts

Regenerative therapy using autologous skeletal myoblasts requires a large number of cells to be prepared for high-level secretion of cytokines and chemokines to induce good regeneration of damaged regions. However, myoblast expansion culture is hindered by a reduction in growth rate owing to cellular quiescence and differentiation, therefore optimization is required. We have developed a kinetic computational model describing skeletal myoblast proliferation and differentiation, which can be used as a prediction tool for the expansion process. In the model, myoblasts migrate, divide, quiesce and differentiate as observed during <i>in vitro</i> culture. We assumed cell differentiation initiates following cell-cell attachment for a defined time period. The model parameter values were estimated by fitting to several predetermined experimental datasets. Using an additional experimental dataset, we confirmed validity of the developed model. We then executed simulations using the developed model under several culture conditions and quantitatively predicted that non-uniform cell seeding had adverse effects on the expansion culture, mainly by reducing the existing ratio of proliferative cells. The proposed model is expected to be useful for predicting myoblast behaviours and in designing efficient expansion culture conditions for these cells

Effect of Rho&ndash;Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

Rho&ndash;associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors of two hiPSC lines (Tic and 1383D2) with different formation of aggregate that attached between single cells in suspension culture. The apparent specific growth rate by long-term exposure to Y-27632, a ROCK inhibitor, was maintained throughout the culture. Long-term exposure to ROCK inhibitor led to an increase in cell division throughout the culture in both lines. Immunofluorescence staining confirmed that hiPSCs forming spherical aggregates showed localization of collagen type I on its periphery. In addition, phosphorylated myosin (pMLC) was localized at the periphery in culture under short-term exposure to ROCK inhibitor, whereas pMLC was not detected at whole the aggregate in culture under long-term exposure. Scanning electron microscopy indicated that long-term exposure to ROCK inhibitor blocked the structural alteration on the surface of cell aggregates. These results indicate that pMLC inhibition by long-term ROCK inhibition leads to enhanced growth abilities of hiPSCs in suspension culture by maintaining the structures of extracellular matrices