Introduction: The manufacture of cell-based products requires assuring sterility through all processes, with aseptic processing in a cleanroom. The environment consists of a critical processing zone (CPZ) that can ensure a level of cleanliness that allows cell culture containers to be opened, and a support zone (SZ) adjacent to it and accessed by an operator. In this study, an environment for cell manufacturing was proposed by designing an air mass balance in an aseptic processing area (APA). Methods: We considered the distribution of particle concentration related to the airflow of clean air passing through a high efficiency particulate air (HEPA) filter and the location of the particle emission sources and set up a model dividing the SZ into two zones vertically: the upper and lower zones in a cleanroom, considering three cases practically. Both the air inlet and outlet were located outside the cleanroom and were connected to the CPZ directly by air ducts (Case 1). The inlets of the CPZ were located in the lower or upper zones of the SZ inside the cleanroom, and the outlets were located in the upper zone (Case 2 or Case 3, respectively). We analyzed how the cleanliness of the APA was affected by different locations of the inlet and outlet of the CPZ by varying the particle emission rate or air change rate. Results: In Case 1, changes in the particle emission rate or air change rate within the SZ did not affect the particle concentration in the CPZ. In Case 2, an increase in the particle emission rate led to an increase in the particle concentration of the CPZ. In Case 3, the particle concentration of the CPZ was not affected by the particle emission rate. Cases 2 and 3 showed differences in particle concentrations between the CPZ and SZ, indicating that the location of the air inlet of the CPZ had an impact on the cleanliness of both zones. The partial circulation of air between the SZ and CPZ exhibited an additional air cleaning effect, leading to a reduction in the particle concentration in the SZ in Cases 2 and 3. Conclusions: These results suggest that the appropriate location of the air inlet and outlet can construct the cleanliness of the APA, which reduces the risk of microbial contamination. In addition, we consider that this approach can realize an APA design policy, which eliminates the need for air ducts between the outside of the cleanroom and the equipment for the CPZ, reduces the requirements for gowning, thereby reducing the required air change rate.

With their many therapeutic functions, mesenchymal stem cells (MSCs) are promising sources for regenerative medicine. However, in the manufacture of MSCs, without a method for exploring the effects of long-term passage on cell proliferation potentials, the design of passage culture processes is challenging. Here, for the process design of the MSC passage culture, we propose a model for predicting the growth rate as a function of the cumulative population doubling level (cPDL) for each passage. Three steps were implemented: (1) passage culture experiments to correlate apparent growth rate with cPDL were conducted, (2) a model for predicting the growth rate as a function of cPDL was developed, and (3) a model to design the passage culture of MSCs from bone marrow (BM-MSCs) and umbilical cord (UC-MSCs) with stochastic simulation was applied. Two design variables (passage number and harvesting time) were investigated to define feasible operation regions as probabilistic design spaces to meet three quality indicators (senescence level, confluency level, and total number of cells) with given probabilities. Consequently, 10 and 62 conditions out of 165 were identified as feasible for BM- and UC-MSCs, respectively, which would contribute to the industrial MSC passage culture process design.

Regenerative medicine is an emerging field of medical treatment characterized by numerous uncertainties, necessitating regulatory frameworks that ensure both patient safety and timely clinical application. Among the three legislative measures established in Japan to facilitate the safety and efficient implementation of regenerative medicine, the Act on the Safety of Regenerative Medicine (RM Safety Act) governs regenerative medicine and cell therapies (RMTs) that utilize processed cells without manufacturing and marketing authorization. These therapies are conducted either as non-commercial clinical trials or as out-of-pocket therapies at the discretion of medical practitioners. More than a decade has passed since the enactment of the RM Safety Act, during which various aspects of RMTs have been clarified. The Evaluation Committee on Regenerative Medicine, Health Sciences Council, published the Summary of the Review of the Act on the Safety of Regenerative Medicine, 5 Years After Its Enforcement, which highlighted key challenges, including the lack of specific regulatory provisions for the storage of cells used in RMTs. In response, the Japanese Society for Regenerative Medicine developed the Basic Points to Consider for Cell Storage under the Act on the Safety of Regenerative Medicine, which outlines guidelines for practitioners intending to store human-derived, specified processed cells and/or their raw materials in a frozen state, as well as for institutions that provides cell storage. This review summarizes the current status and challenges of the RM Safety Act from the perspective of cell storage and provides an overview of the Basic Points to Consider for Cell Storage under the Act on the Safety of Regenerative Medicine and its role as an educational resource.

Mesenchymal stem cells (MSCs) represent a promising route for regenerative medicine because of their therapeutic functions. Considering the anticipated demand growth of MSCs, design spaces (DSs) have been emphasized to ensure cell quality by process design. However, a model that can consider all the effects throughout the multiple steps of MSC manufacturing processes has yet to be presented. Here, we propose a model-based design that considers dynamics and variabilities through the seeding, cultivation, and passage culture of MSCs. An integrated kinetic model was developed, incorporating the effects of seeding heterogeneity and passage-associated senescence into dynamic variations of quality indicators. Two seeding cases were implemented to yield DSs, fulfilling quality specifications with a specified probability. The DS assessment proposed a condition that maximized cell growth efficiency, with confirming robustness against seeding deviations. The presented methodology would contribute to simulation-based decision-making in therapeutic cell manufacturing process design.

This work presents a circular exploration of cryoprotective agents (CPAs) for stem cells using computer-aided molecular design approaches that can comprehensively consider compounds. An exploration cycle was developed that consists of the following five steps: setting conditions, computational evaluation, experimental evaluation, verification experiments, and discussions with experts in biotechnology. It aims to discover promising CPA candidate compounds by incorporating domain knowledge through discussions with the experts. The developed cycle can be applied to fields where the required physical properties have not been clearly known. As a result, 1-methylimidazole and pyridazine were selected as promising CPA candidate compounds, which were both heterocyclic amines. Hence, heterocyclic amines could be a stepping-stone toward the future development of CPAs for stem cells. By repeatedly using the exploration cycle, CPA candidate compounds with better cryoprotective effects could be discovered.

Ultrasound microscopy is the only technique that has the ability to monitor live-cell morphology over a long period of time without causing any damage to the cells, but its longer wavelength prevents one from obtaining high-resolution cell images. Here, we propose a deep-learning (DL) method for generating high-resolution acoustic images. By preparing datasets consisting of many pairs of acoustic and optical-microscope images for the same cells and training them, a high-resolution image comparable to optical microscopy is generated from an acoustic image. Importantly, the most accurate images are generated when three-layer (RGB) images containing not only high-frequency (approximately 180 MHz) images but also lower-frequency (approximately 100 MHz) images are used as the input images, which is attributed to enhanced acoustic absorption in the nucleus because the nucleus resonates in this low-frequency band. The DL scheme with the tri-frequency image input is applied to human mesenchymal stem cells and human induced pluripotent stem cells, and the high image-generation capability is demonstrated. As a result, high-resolution acoustic microscopy images are obtained for the same cells for over 24 h, without the typical cell damage encountered using optical imaging.

Therapies using extracellular vesicles (EVs), exosomes, or cell culture supernatants containing EVs or exosomes (referred to as “EV therapies” in this Guidance) have garnered an increasing amount of interest. However, pharmaceutical products containing EVs as their main active ingredient are yet to receive regulatory approval.The “Basic points to consider regarding the preparation of extracellular vesicles and their clinical applications in Japan,” was announced by the Japanese Society for Regenerative Medicine (JSRM) on March 10, 2021, with a specific focus on exosomes among EVs to promote high safety standards in this evolving field and facilitate the application of EV clinically. The Scientific Committee of the Pharmaceuticals and Medical Devices Agency and the Japanese Society for Extracellular Vesicles have issued reports and statements regarding treatment, resulting in a growing momentum toward treatment in Japan.This article summarized the basic items that should be recognized comprehensively for clinical practice in three categories: (1) risk profiling, (2) preparation (manufacturing) process and quality, and (3) verification of EVs checking items and effectiveness. This guideline will be revised in the future with technological innovations and new findings; nevertheless, it will serve as a guide for the development of treatments.

The colony morphology of induced pluripotent stem cells is deeply related to cell functions. However, three-dimensional information during the growing process remains unexplored. Here, we non-invasively investigate the change in the cross-sectional shape of the colony for a long period of time. We develop a scanning-acoustic-microscopy system to construct spectroscopic acoustic images of cells in culture and then evaluate their height distribution. The images show fringe patterns due to acoustic interference near the edge of the colony, from which the colony height is accurately determined even in the thinner region. The time-variation analysis reveals that the colony shape approaches a dome shape, and the colony grows maintaining the similarity of its cross-sectional shape, and this indicates an exquisite balance between cell migration and cell proliferation in the colony.

In regenerative medicine, mesenchymal stem cells (MSCs) constitute a promising therapeutic route for many diseases. The current quality-by-design guidelines do not clearly define a framework for MSC production. Here, we suggest and experimentally validate a model-based method to determine design spaces (DSs) for MSC cultivation. A kinetic model used in previous work was employed; part of the experimental data was used to re-estimate the maximum specific growth rate in the kinetic model and then calculate the prediction intervals of this parameter. Subsequently, regions of seeding density and harvesting time where both the upper and lower limits of growth predictions met the acceptable number of cells and confluency with given risk levels were defined as DSs. Finally, the established DS was validated with the remaining data; it allowed better predictions of the cell numbers and confluency under specific cultivation conditions and improved the overall robustness of MSC cultivation processes.

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24 h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures. (c) The for All reserved.

This work presents a computational fluid dynamics (CFD) model-based process design of forced convection continuous freezing for human induced pluripotent stem (hiPS) cells considering supercooling of extracellular solutions. The overall model consists of a CFD model for velocity field and heat transfer calculations including the effect of the supercooling, and a cell layer model for cell survival rate calculations. Experimental temperature profiles were obtained using a forced convection-based batch freezer, and the validation of the heat transfer and solidification models was performed. The experiments were carried out by applying a 1.0 K min-1 cooling rate with an inlet velocity of 2.0 m s-1. The model was applied to three different freezer sizes with shelves containing 10, 20 and 30 rows. The applied coolant inlet velocities ranged from 0.50 m s-1 to 4.0 m s-1. It was found that the application of an inlet velocity of 3.0 m s-1 yielded an average cell survival rate of 0.934 with low survival rate heterogeneity in the freezers. This way, the freezing process for hiPS cells could be scaled to up to 170-fold compared to currently used equipment for hiPS cell freezing.

Various techniques have been used for the mechanical stimulation of cells, but most of them require direct contact with cells and can damage them. Here, we propose a focused ultrasound technique for non-invasive localized mechanical stimulation in a culture environment. First, we develop the focused ultrasound system for establishing the spectroscopic images and for applying mechanical stimulation combined with optical observation. Second, we construct the acoustic-absorption spectroscopic images of human mesenchymal stem cells, leading to the finding of the ultrasound absorption band of the nucleus around 130 MHz caused by its resonance. Finally, we find that the nucleus less absorbs ultrasound before the cell division, supporting our view that the acoustic absorption band is related to the resonance of the nucleus. Our results show the potential for realizing selective and effective mechanical stimulation for nuclei non-invasively, which is critically important for a deeper understanding of mechanobiology.

Background & AimHuman induced pluripotent stem cells (hiPSCs) have capability to differentiated into cells of three germ layers, such as endoderm, ectoderm and mesoderm and following various cell lineages. One of the promising application of hiPSCs is the 3D cardiomyocyte differentiation culture to meet the demand of cardiomyocyte production for clinical treatment. We focused on this bioprocess and proposed improved method to control the stability of hiPSCs fate commitment toward cardiomyocytes in batch cultures.Methods, Results & ConclusionIn 3D cardiomyocyte differentiation culture, hiPSCs was forced to make cell aggregates under static culture in 4 days. Cell aggregates were further subjected to a 5 mL bioreactor to conduct suspension culture for 12 days. At the end of 3D cardiac differentiation culture, cell aggregates were dissociated and the cardiomyocytes marker cTnT was detected. As the hallmark of early fate commitment of hiPSCs, the E-cadherin and N-cadherin expression inside cell aggregates were investigated by cryotomy and immunofluorescence staining, and further image processing was implemented to quantifying their expression.cTnT positive ratios showed to be 0.49±0.14 (AVG ±SD) in 15 runs of differentiation cultures, suggesting the processes prone to quality fluctuations. To understand the trigger for the fluctuation, Epithelial-Mesenchymal Transition (EMT) was investigated. By the captured images and following image processing, the cell aggregates were found to exhibited heterogenous localization of E-cadherin and N-cadherin within individual cell aggregate and non-synchronized expression of E-cadherin and N-cadherin among cell aggregates during EMT. To make homogenous and synchronized EMT, we selected the botulinum hemagglutinin (HA), a functional E-cadherin break-up protein, and added to culture when the EMT being triggered. The same 15 runs of differentiation cultures revealed cTnT positive ratio to be 0.49±0.07, providing much less SD in the same AVG, compared to those in conventional culture described above. After the addition of HA, the homogenous and synchronized EMT was observed, suggesting that the spatial heterogeneity of EMT occurred with and the non-synchronized EMT caused the unstable differentiation, leading to quality fluctuation of cTnT in culture process.The robust process in cardiomyocyte differentiation culture was performed through homogenous and synchronized EMT at early satge, which is critical for practical cell manufacturing.

Introduction: Preparing a uniform cell population in high-density seeding of adherent human induced pluripotent stem cells (hiPSC) requires stable culture conditions and consistent culture operation. In this study, we evaluated cell distribution patterns by changing cell seeding operations and their impact on differentiation toward the neuroectodermal lineage. Methods: The hiPSC line 201B7 was seeded at 1.23 x 105 cells/cm2 following a conventional operation, prolongated time of cell seeding suspension or vessel tilting during cell seeding operation. Fluorescent imaging of cell nuclei was performed 24 h following cell seeding and used for spatial heterogeneity analysis. Flow cytometric analysis was also performed seven days after cell differentiation induction toward neuroectodermal lineage. Results: Indices for spatial heterogeneity following high-density cell seeding were proposed to assess cell distribution patterns. Global heterogeneity (HG) was shown to be mostly affected by vessel tilting during cell seeding operation, while local heterogeneity (HL) was affected by prolongated time of cell seeding suspension. Changes in both spatial heterogeneities in the hiPSC population resulted in a lower yield of target neuroectodermal cells compared with the control operation. Conclusion: High-density hiPSC seeding is critical for achieving a higher yield of target cells of neuroectodermal lineage. Understanding the spatial heterogeneity in early stages detects errors in cell culture motion and predicts cell fate in later stages of cell culture. (c) 2024 Japanese Society of Regenerative Medicine. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background & AimThe QbD approach has been spread in pharmaceutical manufacturing. However, it is now known that the difficulties both quality and process exist to indicate the CQAs such as cell potency because of ambiguous drug efficacy with less information for the cellular mode of action and to determine the criticality of PPs because of less robustness of the process.In the present study, we propose an advanced QbD approach to apply cell manufacturing, which will be applicable toward commercial scale. Moreover, in the steps of risk assessment and manufacturing process development, the method to determine the criticality of PPs by considering the control difficulty in the process.Methods, Results & ConclusionThe advanced QbD approach is proposed in cell manufacturing with considering the timing of clinical trials, approval application, and manufacturing process validation as follows. 1. Design of Therapy with QTPP. 2. Design of Product with potential CQAs. 3. Risk Assessment linking MAs and PPs to potential CQAs. 4-1. Manufacturing Process Development and Control Strategy to confirm DS with CMAs and tentative CPPs based on process analytical technology(PAT), 4-2. The preparation of Common Technical Document (CTD) Module 3 (quality) starts, and non-clinical and clinical study start in product validation and verification, confirming the CQA from potential CQAs. 4-3. Product Lifecycle Management with extended DS through PAT for scale-up and approval application. 4-4. Continual Improvement through manufacturing process validation.In addition, we consider the risk assessment linking PPs to potential CQAs by considering the control difficulty in the step 3. 3-1. Make list for whole motions in the specific process. 3-2. Setting the degree of control difficulty in each motion for time, substance amount, momentum, and energy (heat) amount. 3-3. Ranking the degree of control difficulty and pick up the critical motion with a mathematic model. In MSC manufacturing with manual operation (research base), the culture passage process consists of 850 motions, and we extracted two tCPPs for controls of temperature during cell detachment by enzymatic digestion and vessel movement after seeding and in passage process, resulting in the establishment of a semi-automated protocol that involves mathematical decisions.In conclusion, this proposed procedure by considering control difficulties of motions contributes to simplification of extraction of tCPP for potential CQAs.

This work presents an image-based hybrid model incorporating the initial spatial distribution for mesenchymal stem cell (MSC) cultivation process design. First, three levels of seeding bias in static MSC cultivation were experimentally investigated, and the resulting initial distribution was quantified using phase contrast microscopy image analysis. Second, the observed spatial heterogeneity was defined as a new parameter by calculating the standard deviation of the seeded cell number fraction among numerical 8 x 8 tiles on two-dimensional plates. Third, the parameter was incorporated into kinetic models to consider spatial growth limitation. The model was then applied to simulate MSC cultivation processes with experimental data. Using dynamic and stochastic simulation outputs, feasible ranges of cell-harvesting time could be determined to satisfy a given requirement for the minimum cell number and acceptable confluency level. The developed hybrid model could serve as a basis for quantitative decision-making in the design of MSC cultivation processes.

Background & AimThe transplantation of retinal pigment epithelial (RPE) cells derived from human iPS cells has been shown its effectiveness to age-related macular degeneration. However, the process of RPE differentiation takes a few months, and it could make inconsistent between batches. The combination of Yes-associated protein signaling activity which modulates ectodermal differentiation by cell density-dependent and variation in cell density in a vessel from operation might lead to this inconsistency. Therefore, we hypothesized the seeding operation that determines the position of each cell on a culture vessel plays an important role through RPE differentiation.In this research, we aim to stabilize the early phase of RPE differentiation derived from human iPS cells by mechanization of seeding operation.Methods, Results & ConclusionHuman iPS cells (201B7 line) were seeded manually and mechanically on iMatrix-511-coated culture vessels and incubated with differentiation induction medium. At t = 24 h after seeding, two indices of spatial heterogeneity of cell density (local and global heterogeneity) were calculated by using nuclear stained images. In brief, local and global heterogeneity indicate the variation within each locality and the variation overall a culture vessel, respectively. The cells were collected on day 7 of differentiation and analyzed by flow cytometry to evaluate the ratio of neural ectoderm (Target lineage) and epidermal ectoderm (Non-target lineage).Based on spatial heterogeneity analysis, variation of global heterogeneity in machinery operation was lower than in manual operation. Then, the mean value of local heterogeneity in machinery operation was higher than in manual operation. Based on flow cytometric analysis, variation of the ratio of neural ectoderm and epidermal ectoderm in machinery operation was lower than manual operation. Moreover, the yield of neural ectoderm was improved by mechanization. It was suggested that the control of spatial heterogeneity by mechanization stabilized the early phase of RPE differentiation.In conclusion, we demonstrated that stabilization of the early phase of RPE differentiation derived from human iPS cells by mechanization of seeding operation.

Human induced pluripotent stem (hiPS) cells have demonstrated promising potential in regenerative medical therapeutics. After successful clinical trials, the demand for hiPS cells has steadily increased. Therefore, the optimization of hiPS cell freezing processes for storage and transportation is essential. Here, we presented a computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for hiPS cells. This study was based on a model that calculates cell survival rates after thawing, and the model was extended to evaluate cell potentials until 24 h after seeding. To estimate parameter values for this extension, freezing experiments were performed using constant cooling rates. Using quality and productivity indicators, we evaluated 16,206 temperature profiles using our model, and a promising profile was obtained. Finally, an experimental investigation of the profile was undertaken, and the contribution of the temperature profile to both quality and productivity was confirmed.

Background & AimMesenchymal stem cells (MSCs) have become one of the candidates for regenerative medicine. Such cells have a multi-lineage differentiation ability, can modulate immune responses, and can also promote tissue regeneration through the secretome.As the number of MSCs isolated from the tissues is much less than the number of cells needed for treatment, an in vitro expansion process is necessary. Microcarriers (MCs), which are a class of small beads is used as a substrate to prepare suspension cultures and have been recognized as a potential tool for cost-effective large-scale cell expansion process due to providing a larger surface area per unit volume compared to planar cultures.Seeding is the first step of the expansion process to perform cell attachment on MCs. Quantity, quality and stability are required for a considerable seeding process. By increasing microcarrier concentration, CMC (cm2/mL), the cell attachment efficiency of bioreactor showed an increasing trend and finally plateaued in a static seeding condition. While at the same time, we observed a decreasing trend of apparent specific growth rate. Hence, we began to consider the heterogeneity of cell attachment on single MC as a crucial factor of stability.Methods, Results & ConclusionMSCs were seeding on MC with lower CMC condition (3.84 cm2/mL) or higher CMC condition (23.04 cm2/mL) and static or dynamic condition. After 24 hours, 3-dimensional images of MCs and MSCs labelled by fluorescence with nuclei were analyzed to calculate the cell attachment efficiency on MC, αMC (-). Three batches of each seeding condition were performed for the reproducibility. Increasing of the CMC improve the average cell attachment efficiency of MC. (Figure 1) The distribution of αMC in the lower CMC condition had less fluctuation than higher CMC condition indicated by the box size. In lower CMC condition, the dynamic condition was preferred as the static condition had a peak skewed to the 0.Our research provided a method to analysis the stability of seeding process by heterogeneity. We found that the heterogeneity of seeding would influence by the CMC and mixing operation which are suggested to consider during development of seeding process.