Masahiro Kino-oka

Osaka University | Handai · Department of Biotechnology

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...

Human induced pluripotent stem cells (hiPSCs) are an attractive cell source for regenerative medicine. For its widespread use as a starting material, a robust storage and distribution system in the frozen state is necessary. For this system, managing transient warming during storage and transport is essential, but how transient warming affects cell...

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 super...

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 neuroectoderm...

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...

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 explo...

Various techniques have been used for mechanical stimulation on cells, but most of them require direct contact with cells and can damage them. Here, we propose focused ultrasound technique for non-invasive localized mechanical stimulation in culture environment. First, we develop the focused ultrasound system for establishing the spectroscopic imag...

Although the potential of stem cells to differentiate into several cell types has shown promise in regenerative medicine, low differentiation efficiency and poor reproducibility significantly limit their practical application. We developed an effective and robust differentiation strategy for the efficient and robust generation of neural progenitor...

Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchrono...

Introduction Understanding the critical factors for the maturation of human induced pluripotent stem cell (hiPSC)-derived cardiac tissue is important for further development of culture techniques. Rotating flow culture, where the tissues float in the culture medium by balancing its gravitational settling and the medium flow generated in rotating di...

The ability of mesenchymal stem cells (MSCs) to synthesize and degrade extracellular matrix (ECM) is important for MSC-based therapies. However, the therapeutic effects associated with ECM remodeling in cultured MSCs have been limited by the lack of a method to assess the ability of cultured cells to degrade ECM in vitro. Here, we describe a simple...

This manuscript discusses the role of process systems engineering (PSE) in ensuring quality manufacturing of Mesenchymal Stem Cells (MSCs) through seeding, cultivation, and stabilization. A comparison of the MSC production to small molecules and therapeutic protein production processes illustrated the leap in complexity in ensuring quality manufact...

Background Human induced pluripotent stem cells (hiPSCs) provide an in vitro system to identify the impact of cell behavior on the earliest stages of cell fate specification during human development. Here, we developed an hiPSC-based model to study the effect of collective cell migration in meso–endodermal lineage segregation and cell fate decision...

Living cells sense various mechanical stimuli and respond to them by adjusting their tissue morphogenesis, self-renewal, and differentiation. Because stem cells seem to be more sensitive to the stimuli, many studies have been conducted to control their behavior. However, many of these mechanisms remain unexplored, because stimulus extends throughou...

Cell sheet engineering, a scaffold-free tissue fabrication technique, has proven to be an important breakthrough technology in regenerative medicine. Over the past two decades, the field has developed rapidly in terms of investigating fabrication techniques and multipurpose applications in regenerative medicine and biological research. This review...

Current seeding processes of mesenchymal stem cells (MSC) in 2D in vitro culture systems rely on the experience and practice of experimentalists. A systematic understanding of the operation of this process is needed especially considering future scale-out and automation. This work presents a computational fluid dynamics (CFD) model-based design of...

In recent years, extracellular vesicles (EVs)11 Extracellular vesicles; the positioning paper of the International Society of Extracellular Vesicles (ISEV) (Minimal Information for Studies of Extracellular Vesicles [MISEV] 2018, Journal of Exarcellular Vesicles [JEV]; Updating MISEV, 2021, JEV) states that authors are urged to consider use of opera...

Introduction Adipose-derived stem cells (ASCs) secrete various growth factors to promote wound healing and to regenerate various tissues, such as bone, cartilage, and fat tissue. Subcutaneous adipose tissue is a considerable cell source in clinical practice and can be collected relatively easily and safely under local anesthesia. Moreover, platelet...

Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E‐cadherin function‐blocking agent...

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in response to biomechanical microenvironmen...

Rho–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)...

Recent developments for the manufacturing of cell-based products have focused on the advancement of products to clinical trials or commercialization, with awareness of the importance of cost-based effectiveness in cell manufacturing. The mechanization of cell-processing operations is advantageous for the reproducibility and stability of product qua...

The filling process is a part of downstream processing in cell manufacturing. In this study, volume (V/VT), particle density (D/D0), and cell potential (P/P0) quality indices were proposed for the outputs of the filling process designed for human-induced pluripotent stem cell products. Instability in the filling process was evaluated by using an al...

This work presents a multilayered model-based approach to scale-up slow and forced convection-based freezing of human induced pluripotent stem cells. The approach combines a hybrid single-cell freezing model (covering the cell and cryovial layers) with a computational fluid dynamics model (covering the freezer layer). The proposed approach can calc...

This work presents a hybrid-model-based design of a fill-freeze-thaw processes for human induced pluripotent stem (hiPS) cells. For freezing, we applied our previously presented single-cell model, which describes heat transfer, dehydration, and crystallization during freezing. New models have been developed for filling and thawing. The accumulation...

This work details the development of a five-step workflow for design space determination of mesenchymal stem cell (MSC) cultivation. The workflow employs mechanistic models to represent system dynamics of MSC cultivation processes while introducing stochastic simulations to capture uncertainties. The developed workflow was applied to an MSC cultiva...

The cover image is based on the Review Mechanobiological conceptual framework for assessing stem cell bioprocess effectiveness by Mee‐Hae Kim and Masahiro Kino‐oka., https://doi.org/10.1002/bit.27929.

The manufacturing of regenerative medicine product consists of variety of processes in upstream, downstream, and outer-stream. Features inherent in cell processing cause the fluctuation of process and the instability of final product quality, requiring a novel doctrine. This article describes the features of cell processing as well as product quali...

Fully realizing the enormous potential of stem cells requires developing efficient bioprocesses and optimizations founded in mechanobiological considerations. Here, we emphasize the importance of mechanotransduction as one of the governing principles of stem cell bioprocesses, underscoring the need to further explore the behavioral mechanisms invol...

Understanding the cellular behavioral mechanisms underlying memory formation and maintenance in human induced pluripotent stem cell (hiPSC) culture provides key strategies for achieving stability and robustness of cell differentiation. Here, we show that changes in cell behavior-driven epigenetic memory of hiPSC cultures alter their pluripotent sta...

Industrial/commercial-scale biological applications including cell therapy, drug testing and regenerative medicine, require the production of a large number of cells. A suspension culture method has been extensively used to generate sufficiently large quantities of high-quality cells in a defined form. In this study, we carried out the present nume...

The dynamic migratory behavior of human mesenchymal stem cells (hMSCs) has a significant impact on the epigenetic profiles that determine fate choice and lineage commitment during differentiation. Here we report a novel approach to enhance repeated migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer surfac...

An orbitally shaken bioreactor is the most popular system for cell cultivation in a suspension culture owing to its unique characteristics. However, conventional bioreactors struggle to meet the anticipated demand in practical applications due to their limitations. In this study, numerical simulations are conducted to evaluate the performance of a...

The realization of the enormous potential of stem cells requires development of efficient bioprocesses and optimization drawing drawn from mechanobiological considerations. Here, we emphasize the importance of mechanotransduction as one of the governing principles of stem cell bioprocesses, underscoring the need to further explore the behavioral me...

Bubble sparging has been used to supply oxygen to large-scale bioreactor systems. However, sparged bubbles cause cell death by rupturing due to shear stress, and the foam layer carries a risk of contamination. Large-scale culture of human induced pluripotent stem cells (hiPSCs) is required for manufacturing, but hiPSCs show high sensitivity to shea...

The application of induced pluripotent stem (iPS) cells holds significant promise for regenerative medicine and drug screening. To produce sufficient quantities of high-quality iPS cells in undifferentiated form, a wave bioreactor has been widely used for static cell cultivation. A systematic optimization of shear stress and cell dispersion has bee...

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscl...

Induced pluripotent stem cells (iPSCs) are considered to be ideal and promising cell sources for various applications such as regenerative medicine and drug screening. However, effective mass production systems for the stable supply of desired numbers of iPSCs are yet to be developed. This review introduces the various approaches that are currently...

This work presents a model-based assessment of temperature profiles in slow freezing for human induced pluripotent stem (hiPS) cells. It is based on our single-cell model that can quantify temperature distribution in a vial, cell volume change through transmembrane water transport, and intracellular ice formation during freezing. This mechanistic m...

Three‐dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contribu...

The cover image is based on the Original Article Apoptosis‐based method for determining lot sizes in the filling of human‐induced pluripotent stem cells by Hirokazu Sugiyama et al., https://doi.org/10.1002/term.3127.

During culture with feeder cells, deviation from the undifferentiated state of human induced pluripotent stem cells (hiPSCs) occurs at a very low frequency. Anomalous cell migration in central and peripheral regions of hiPSC colonies has been suggested to be the trigger for this phenomenon. To confirm this hypothesis, sequential cell migration prio...

Skeletal muscle comprises a heterogeneous population of myoblasts and fibroblasts. Autologous skeletal muscle myoblasts are transplanted to patients with ischemia to promote cardiac regeneration. In damaged hearts, various cytokines secreted from the skeletal muscle myoblasts promote angiogenesis and consequently the recovery of cardiac functions....

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 apop...

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...

In order to practically apply induced pluripotent stem (iPS) cells to regenerative medicine, a large amount of undifferentiated iPS cells should be produced by using an automated/scaled-up suspension culture system. However, in large-scale culture, oxygen supply to iPS cells away from the gas-liquid interface can be insufficient. In this numerical...

Background & Aim Aseptic processing is one of the most important issues in the production of cell-based products which are handled by opening the culture vessels. The practical level of aseptic processing depends on the design of aseptic environment where the equipment for research are installed in the cell manufacturing facility. An aseptic enviro...

The critical issues facing stem cell bioprocess development today include the establishment of bioengineering principles and design concepts that can deliver reproducible and robust manufacturing processes. The principles for building and nurturing culture environment, operation, and human resources as well as developing automated system can be use...

The cover image is based on the Review Article Designing a blueprint for next‐generation stem cell bioprocessing development by Mee‐Hae Kim and Masahiro Kino‐oka, https://doi.org/10.1002/bit.27228.

Understanding of the fundamental mechanisms of epigenetic modification in the migration of human mesenchymal stem cells (hMSCs) provides surface design strategies for controlling self-renewal and lineage commitment. We investigated the mechanism underlying muscle lineage switching of hMSCs by cellular and nuclear deformation during cell migration o...

Understanding how defects in mechanotransduction affect cell‐to‐cell variability will add to our fundamental knowledge of human pluripotent stem cell (hPSC) culture, and may suggest new approaches for achieving a robust, reproducible, and scalable process that results in consistent product quality and yields. Here, we review the current state of ou...

Abstarct This work presents a model-based design of slow freezing processes for human induced pluripotent stem (hiPS) cells considering quality variation inside a container. A single-cell model was developed that integrated heat transfer, mass transfer, and crystallisation models. These mechanistic models described the radial and temporal temperatu...

This work presents multiobjective and dynamic optimization of temperature profile for the slow freezing of human induced pluripotent stem (hiPS) cells. A single-cell model was developed that can quantify (i) temperature distribution in a container that causes intracontainer conditional variation, (ii) cell volume change through transmembrane water...

Understanding of kinetics on aggregate behaviors of human induced pluripotent stem cells (hiPSCs) is critical knowledge for culture design because aggregate behaviors are considered to affect cell growth. In this study, we elucidated kinetics on aggregate behaviors of two types of hiPSCs (253G1 and 201B7 lines) to clarify the influence of aggregate...

To scale-up a filling process in a cell manufacturing system, the impact of the processing time on the final product quality should be evaluated. It is known that the suspended cells in a cryopreservation solution are subjected to various types of stress factors, which eventually results in apoptotic or necrotic death. In this study, the aim was to...

The creation of a blueprint for stem cell bioprocess development that it is easily readable and shareable among those involved in the construction of the bioprocess is a necessary step toward full‐fledged bioprocess integration. The blueprint provides the culturing tools and methodologies, designed to highlight knowledge gaps within biological scie...

The manufacturers of cell therapy and regenerative medicine products must design manufacturing operations to deliver the required level of stability. Process consistency, maintenance of the aseptic environment (to prevent contamination), assurance of line separation (to prevent cross-contamination and operational confusion), containment (for avoida...

Human mesenchymal stem cells (hMSCs) are considered to be able to adapt to environmental changes induced by gravity during cell expansion. In this study, we investigated neurogenic differentiation potential of passaged hMSCs under conventional gravity and simulated microgravity conditions. Immunostaining, quantitative RT-PCR, and western blot analy...

Cells sense and respond to environmental changes induced by gravity. Although reactions to conventional culture have been intensively studied, little is known about the cellular reaction to simulated microgravity conditions. Thus, in this study, we investigated the effects of simulated microgravity on human mesenchymal stem cells using a three-dime...

Applications of human induced pluripotent stem cell (hiPSC) culture are impaired by problems with long term maintenance of pluripotency. In this study, we report that exposure to botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, suppressed deviation from an undifferentiated state in hiPSC colonies. Time-lapse imaging of live cell...

Introduction: Toward the commercial production of human-induced pluripotent stem (hiPS) cells, the process design and operation have to be standardized. Considering the change in cell quality during filling of the hiPS cells into containers, lot sizing during filling also needs to be standardized. Methods: We present a distribution-based approac...

Background & Aim When the production of cell-based products in the container handling which is handled by opening the vessels, most of cell processing facilities, which install commercial isolators, are difficult to be operated with high availability. The main steps of the cell production, such as medium exchange or passaging operations, must work...

Preventing the contamination of processed cells is required for achieving reproducible manufacturing. A droplet is one of the potential causes contamination in cell manufacturing. The present study elucidates the formation mechanism and characteristics of droplets based on the observation and detection of droplets on the base surface of the biologi...