Computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for human induced pluripotent stem cells.

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.

Novel strategy to improve hepatocyte differentiation stability through synchronized behavior-driven mechanical memory of iPSCs.

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, to synchronize behavior-driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration-and time-dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes-associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.

The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells.

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 contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.

Mechanobiological conceptual framework for assessing stem cell bioprocess effectiveness.

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 involved in sensing mechanical cues and coordinating transcriptional responses. We identify the sources of intrinsic, extrinsic, and external noise in bioprocesses requiring further study, and discuss the criteria and indicators that may be used to assess and predict cell-to-cell variability resulting from environmental fluctuations. Specifically, we propose a conceptual framework to explain the impact of mechanical forces within the cellular environment, identify key cell state determinants in bioprocesses, and discuss downstream implementation challenges.

Designing a blueprint for next-generation stem cell bioprocessing development.

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 sciences and bioengineering. This review highlights a blueprint for stem cell bioprocessing development using a landscape architecture approach that can aid the development of culture technologies and tools that satisfy the demands for stem cell-derived products for use in clinical and industrial applications. This work is intended to provide insights to cell biologists, geneticists, bioengineers, and clinicians seeking knowledge outside of their field of expertise and fosters a leap from a reductionist approach to one, that is, globally integrated in stem cell bioprocessing.

Maintenance of human chondrogenic phenotype on a dendrimer-immobilized surface for an application of cell sheet engineering.

BACKGROUND: Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets. RESULTS: During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers. CONCLUSIONS: The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.

Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 106 cells ml-1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture.

A Novel Strategy for Simple and Robust Expansion of Human Pluripotent Stem Cells Using Botulinum Hemagglutinin.

Clinical and industrial application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust strategies to sustain cultures in an undifferentiated state. Here, we describe a simple and robust method to culture and propagate hPSCs, which we anticipate will remove major roadblocks in investigating the basic properties of undifferentiated hPSCs and accelerate cell-based manufacturing. We also provide an overview of the use of botulinum hemagglutinin, an inhibitor of E-cadherin, to maintain and expand various hPSC lines in an undifferentiated state in different culture conditions. Hemagglutinin selectively removes cells that have lost the undifferentiated state, dissociates aggregates in situ, and is easy to use, scalable, and reproducible.

Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface.

OBJECTIVES: To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis. RESULTS: hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium. CONCLUSIONS: Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture.

Human induced pluripotent stem cells (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 the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 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 the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

Kinetic analysis of deviation from the undifferentiated state in colonies of human induced pluripotent stem cells on feeder layers.

Understanding of the fundamental mechanisms that govern unintentional differentiation of human induced pluripotent stem cells (hiPSCs) provides key strategies to maintain their undifferentiated state during cell expansion. This study focused on deviation from the undifferentiated state in hiPSC colonies during culture with feeder cells. Deviated cells from the undifferentiated state of hiPSCs in cultures with SNL and MEF feeder cells were observed at the center and periphery of the colonies, respectively, accompanied by dramatic changes in the cell morphology from small to large flattened shapes. It was found that the deviation of undifferentiated hiPSCs in culture with SNL feeder cells caused deviated cells in the center of the colony through spontaneous occurrence in a colony size-dependent manner, whereas the deviation of undifferentiated hiPSCs in culture with MEF feeder cells caused deviated cells in the periphery of the colonies through accidental events during migration in a colony size-independent manner. Based on a kinetic analysis of time-lapse images of single hiPSC colonies, the specific growth rate for replication of deviated cells from the undifferentiated state in culture with SNL feeder cells was 1.83 and 3.57 times higher than those of undifferentiated cells and transformation, respectively, meaning that the deviation of undifferentiated hiPSCs dramatically expanded through replication of deviated cells from the undifferentiated state and transformation once deviation from the undifferentiated state had occurred. In the case of MEF feeder cells, the specific growth rates for replication of deviated cells from the undifferentiated state was 3.12 times higher than that of undifferentiated cells, whereas the rate by transformation exhibited a negligible level compared with the rates of replication for undifferentiated cells and deviated cells from undifferentiated state, meaning that deviation of undifferentiated hiPSCs dramatically expanded only through replication of deviated cells from the undifferentiated state. These results suggest that once deviation has occurred in a colony, the deviated cells from undifferentiated state undertake dramatic invasion to occupy the colony. Maintenance of the undifferentiated state in subcultures inevitably requires vigilant care to remove any colonies that include deviated cells from the undifferentiated state.

A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors.

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 synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1+ and NKX6.1+ pancreatic progenitor cells in high yields. The PDX1+ and PDX1+ /NKX6.1+ cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.

Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin.

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.

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage.

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10(5) cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.

Endothelial cell behavior inside myoblast sheets with different thickness.

Using a cell sheet stacking method, we developed an in vitro culture system in which green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) were cultured under human skeletal muscle myoblast (HSMM) sheets with different layer numbers. Our aim in developing this system was to examine the different endothelial behaviors in the cell sheet. During 96 h of incubation, in monolayer HSMM sheet, HUVECs quickly reached the top of the cell sheet and detached. In three-layered HSMM sheet, HUVECs also migrated to the top layer and formed island-shaped aggregates. In five-layered HSMM sheet, HUVECs migrated into the middle of the cell sheet and formed net-shaped aggregates. In seven-layered HSMM sheet, HUVECs migrated in the basal of the cell sheet and formed sparse net-shaped aggregates. The thickness of the HSMM sheet, which can be controlled by the layer number of the cell sheet, is therefore an important parameter that affects the migration time, encounters, localization, and morphology of HUVECs inside the HSMM sheet.

Stable and efficient generation of functional iPSC-derived neural progenitor cell rosettes through regulation of collective cell-cell behavior.

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 cell rosettes from induced pluripotent stem cells (iPSCs) incorporating botulinum hemagglutinin (HA). Treatment with HA suppressed the spontaneous differentiation of iPSCs cultured under undirected differentiation conditions, resulting in the preservation of their pluripotency. Moreover, treatment with HA during neural progenitor differentiation combined with dual SMAD inhibition generated a highly homogeneous population of PAX6-and SOX1-expressing neural progenitor cells with 8.4-fold higher yields of neural progenitor cells than untreated control cultures. These neural progenitor cells formed radially organized rosettes surrounding the central lumen. This differentiation method enhanced the generation of functional iPSC-derived neural progenitor cell rosettes throughout the culture vessel, suggesting that the regulation of collective cell-cell behavior using HA plays a morphogenetically important role in rosette formation and maturation. These findings show the significance of HA in the suppression of spontaneous differentiation through spatial homogeneity. The study proposes a novel methodology for the efficient derivation of functional iPSC-derived neural progenitor cell rosettes.

An in vitro culture platform to study the extracellular matrix remodeling potential of human mesenchymal stem cells.

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 in vitro culture platform for studying the ECM remodeling potential of cultured MSCs using a high-density collagen (CL) surface. Cells on the CL surface have remarkable ability to degrade collagen fibrils by secreting matrix metalloproteinase (MMP); to study this, the marker collagen hybridizing peptide (CHP) was used. Confirming the ECM remodeling potential of MSCs with different population doublings (PDs), young and healthy γ-H2AX-negative cells, a marker of DNA damage and senescence, showed more extensive collagen degradation on the CL surface, whereas damaged cells of γ-H2AX-positive cells showed no collagen degradation. The frequency of γ-H2AX-/CHP + cells at PD = 0 was 49%, which was 4.9-fold higher than that at PD=13.07, whereas the frequency of γ-H2AX+/CHP- at PD=13.07 was 50%, which was 6.4-folds higher than that at PD=0. Further experimentation examining the in vitro priming effect of MSCs with the pro-inflammatory cytokine interferon-γ treatment showed increased frequency of cells with ECM remodeling potential with higher MMP secretion. Thus, this culture surface can be used for studying the ECM remodeling capacity of ex vivo-expanded MSCs in vitro and may serve as a platform for prediction in vivo ECM remodeling effect. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) remodeling potential of cultured mesenchymal stem cells (MSCs) is important for assessing the effectiveness of MSC-based therapy. However, methods to assess the ability of cultured cells to degrade ECM in vitro are still lacking. Here, we developed a simple in vitro culture platform to study the ECM remodeling potential of cultured MSCs using high-density collagen surfaces. This platform was used to evaluate the ECM remodeling potential of long-term ex vivo-expanded MSCs in vitro.

An in vitro culture platform for studying the effect of collective cell migration on spatial self-organization within induced pluripotent stem cell colonies.

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 decisions through the control of space confinement using a detachable ring culture system. RESULTS: The actomyosin organization of cells at the edge of undifferentiated colonies formed in a ring barrier differed from that of the cells in the center of the colony. In addition, even in the absence of exogenous supplements, ectoderm, mesoderm, endoderm, and extraembryonic cells differentiated following the induction of collective cell migration at the colony edge by removing the ring-barrier. However, when collective cell migration was inhibited by blocking E-cadherin function, this fate decision within an hiPSC colony was altered to an ectodermal fate. Furthermore, the induction of collective cell migration at the colony edge using an endodermal induction media enhanced endodermal differentiation efficiency in association with cadherin switching, which is involved in the epithelial-mesenchymal transition. CONCLUSIONS: Our findings suggest that collective cell migration can be an effective way to drive the segregation of mesoderm and endoderm lineages, and cell fate decisions of hiPSCs.

Recent Advances in Cell Sheet Engineering: From Fabrication to Clinical Translation.

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 highlights the most important achievements in cell sheet engineering to date. We first discuss cell sheet harvesting systems, which have been introduced in temperature-responsive surfaces and other systems to overcome the limitations of conventional cell harvesting methods. In addition, we describe several techniques of cell sheet transfer for preclinical (in vitro and in vivo) and clinical trials. This review also covers cell sheet cryopreservation, which allows short- and long-term storage of cells. Subsequently, we discuss the cell sheet properties of angiogenic cytokines and vasculogenesis. Finally, we discuss updates to various applications, from biological research to clinical translation. We believe that the present review, which shows and compares fundamental technologies and recent advances in cell engineering, can potentially be helpful for new and experienced researchers to promote the further development of tissue engineering in different applications.

Effect of morphological change on the maturation of human induced pluripotent stem cell-derived cardiac tissue in rotating flow culture.

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 disk-shaped culture vessels, is one of culture systems used for tissue engineering. It has previously been demonstrated that rotating flow culture leads to the formation of matured cardiac tissue with higher levels of function and structure than the other culture systems. However, the detailed mechanisms underlying the maturation of cardiac tissue remain unclear. This study investigated the maturation process of hiPSC-derived cardiac tissue in rotating flow culture with a focus on morphological changes in the tissue, which is a trigger for maturation. Methods: The cardiac tissue, which consisted of cardiomyocytes derived from hiPSCs, was cultured on the 3D scaffold of poly (lactic-co-glycolic) acid (PLGA)-aligned nanofibers, in rotating flow culture for 5 days. During the culture, the time profile of projected area of tissue and formation of maturation marker proteins (ß-myosin heavy chain and Connexin-43), tissue structure, and formation of nuclear lamina proteins (Lamin A/C) were compared with that in static suspension culture. Results: The ratio of the projected area of tissue significantly decreased from Day 0 to Day 3 due to tissue shrinkage. In contrast, Western blot analysis revealed that maturation protein markers of cardiomyocytes significantly increased after Day 3. In addition, in rotating flow culture, flat-shaped nuclei and fiber-like cytoskeletal structures were distributed in the surface region of tissue where medium flow was continuously applied. Moreover, Lamin A/C, which are generally formed in differentiated cells owing to mechanical force across the cytoskeleton and critically affect the maturation of cardiomyocytes, were significantly formed in the tissue of rotating flow culture. Conclusions: In this study, we found that spatial heterogeneity of tissue structure and tissue shrinkage occurred in rotating flow culture, which was not observed in static suspension culture. Moreover, from the quantitative analysis, it was also suggested that tissue shrinkage in rotating flow culture contributed its following tissue maturation. These findings showed one of the important characteristics of rotating flow culture which was not revealed in previous studies.