Yuki Kagawa

Modeling the nutrient removal process in aerobic granular sludge system by coupling the reactor- and granule-scale models

We developed a model for nutrient removal in an aerobic granular sludge system. This model can quantitatively describe the start‐up of the system by coupling a model for studying the population dynamics of the granules in the reactor (reactor‐scale model) and a model for studying the microbial community structure in the granules (granule‐scale model). The reactor‐scale model is used for simulation for 10 days from the start, during which the granule size is relatively small; the granule‐scale model is used after Day 10. The present approach proposes the output data of the reactor‐scale model after 10 days as initial conditions for the granule‐scale model. The constructed model satisfactorily describes experimental data in various spatial and temporal scales, which were obtained in this study by performing the anaerobic–aerobic–anoxic cycles using a sequencing batch reactor. Simulations using this model quantitatively predicted that the stability of nutrient removal process depended largely on the dissolved oxygen (DO) concentration, and the DO setpoint adaptation could improve the nutrient removal performance. Biotechnol. Bioeng. 2015;112: 53–64.

Modeling of stem cell dynamics in human colonic crypts in silico

BackgroundSeveral possible scenarios of cellular dynamics in human colonic crypts have been inferred from transgenic animal experiments. However, because of the discrepancy in size and physiology between humans and animals, quantitative predictions of tissue renewal and cancer development are difficult to execute.MethodsA two-dimensional individual based model was developed for the first time to predict cellular dynamics in human colonic crypts. A simple scenario, in which stem cells were not fixed positionally, divide symmetrically and asymmetrically in a stochastic fashion in the lower part of the crypt, was proposed and implemented in the developed model. Numerical simulations of the model were executed in silico.ResultsBy comparing the results of computational simulations with available experimental data, the presented scenario was consistent with various experimental evidence. Using this scenario, we simulated and visualized monoclonal conversion in the human colonic crypt. We also predicted that the propensity for monoclonal expansion of a mutant cell was largely dependent on the phenotype, the cell type, the position and the state of the crypt.ConclusionsUsing the computational framework developed in this study, model users can verify possible scenarios of stem cell dynamics occurring in human colonic crypts and quantitatively predict cell behavior. Its applicability in scenario verification and predictability makes it a valuable tool for elucidation of stem cell dynamics in human colonic crypts.

Oxygen consumption of human heart cells in monolayer culture

Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71±0.38pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77±0.32pmol/h/cell and 1.61±0.70pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart.

Mathematical modeling of dormant cell formation in growing biofilm.

Understanding the dynamics of dormant cells in microbial biofilms, in which the bacteria are embedded in extracellular matrix, is important for developing successful antibiotic therapies against pathogenic bacteria. Although some of the molecular mechanisms leading to bacterial persistence have been speculated in planktonic bacterial cell, how dormant cells emerge in the biofilms of pathogenic bacteria such as Pseudomonas aeruginosa remains unclear. The present study proposes four hypotheses of dormant cell formation; stochastic process, nutrient-dependent, oxygen-dependent, and time-dependent processes. These hypotheses were implemented into a three-dimensional individual-based model of biofilm formation. Numerical simulations of the different mechanisms yielded qualitatively different spatiotemporal distributions of dormant cells in the growing biofilm. Based on these simulation results, we discuss what kinds of experimental studies are effective for discriminating dormant cell formation mechanisms in biofilms.

Direct measurement of local dissolved oxygen concentration spatial profiles in a cell culture environment

Controlling local dissolved oxygen concentration (DO) in media is critical for cell or tissue cultures. Various biomaterials and culture methods have been developed to modulate DO. Direct measurement of local DO in cultures has not been validated as a method to test DO modulation. In the present study we developed a DO measurement system equipped with a Clark‐type oxygen microelectrode manipulated with 1 μm precision in three‐dimensional space to explore potential applications for tissue engineering. By determining the microelectrode tip position precisely against the bottom plane of culture dishes with rat or human cardiac cells in static monolayer culture, we successfully obtained spatial distributions of DO in the medium. Theoretical quantitative predictions fit the obtained data well. Based on analyses of the variance between samples, we found the data reflected “local” oxygen consumption in the vicinity of the microelectrode and the detection of temporal changes in oxygen consumption rates of cultured cells was limited by the diffusion rate of oxygen in the medium. This oxygen measuring system monitors local oxygen consumption and production with high spatial resolution, and can potentially be used with recently developed oxygen modulating biomaterials to design microenvironments and non‐invasively monitor local DO dynamics during culture. Biotechnol. Bioeng. 2015;112: 1263–1274.

Thicker three-dimensional tissue from a “symbiotic recycling system” combining mammalian cells and algae

In this paper, we report an in vitro co-culture system that combines mammalian cells and algae, Chlorococcum littorale, to create a three-dimensional (3-D) tissue. While the C2C12 mouse myoblasts and rat cardiac cells consumed oxygen actively, intense oxygen production was accounted for by the algae even in the co-culture system. Although cell metabolism within thicker cardiac cell-layered tissues showed anaerobic respiration, the introduction of innovative co-cultivation partially changed the metabolism to aerobic respiration. Moreover, the amount of glucose consumption and lactate production in the cardiac tissues and the amount of ammonia in the culture media decreased significantly when co-cultivated with algae. In the cardiac tissues devoid of algae, delamination was observed histologically, and the release of creatine kinase (CK) from the tissues showed severe cardiac cell damage. On the other hand, the layered cell tissues with algae were observed to be in a good histological condition, with less than one-fifth decline in CK release. The co-cultivation with algae improved the culture condition of the thicker tissues, resulting in the formation of 160 μm-thick cardiac tissues. Thus, the present study proposes the possibility of creating an in vitro “symbiotic recycling system” composed of mammalian cells and algae.

Stepping motion of the organelle in a perfused characean cell

The sliding motion of organelles in a perfused characean internodal cell has been investigated. A small number of myosin molecules attached to the organelle are known to slide along actin bundles lying on the inner surface of the cell. The stepping motion of the organelles was first observed using a high‐speed camera with a time resolution of 1 ms. The spatial frequency of all pairwise differences in displacement of the stepping motion has been estimated to be 0.012–0.014 nm−1, which implies that the organelle moves with large steps of 71–83 nm along the actin bundles in a perfused characean cell.

Chaotic behavior in the locomotion of Amoeba proteus

SummaryThe locomotion ofAmoeba proteus has been investigated by algorithms evaluating correlation dimension and Lyapunov spectrum developed in the field of nonlinear science. It is presumed by these parameters whether the random behavior of the system is stochastic or deterministic. For the analysis of the nonlinear parameters, n-dimensional time-delayed vectors have been reconstructed from a time series of periphery and area ofA. proteus images captured with a charge-coupled-device camera, which characterize its random motion. The correlation dimension analyzed has shown the random motion ofA. proteus is subjected only to 3–4 macrovariables, though the system is a complex system composed of many degrees of freedom. Furthermore, the analysis of the Lyapunov spectrum has shown its largest exponent takes positive values. These results indicate the random behavior ofA. proteus is chaotic and deterministic motion on an attractor with low dimension. It may be important for the elucidation of the cell locomotion to take account of nonlinear interactions among a small number of dynamics such as the sol-gel transformation, the cytoplasmic streaming, and the relating chemical reaction occurring in the cell.

Real-time quantitation of internal metabolic activity of three-dimensional engineered tissues using an oxygen microelectrode and optical coherence tomography

Recent progress in tissue engineering technology has enabled us to develop thick tissue constructs that can then be transplanted in regenerative therapies. In clinical situations, it is vital that the engineered tissues to be implanted are safe and functional before use. However, there is currently a limited number of studies on real-time quality evaluation of thick living tissue constructs. Here we developed a system for quantifying the internal activities of engineered tissues, from which we can evaluate its quality in real-time. The evaluation was achieved by measuring oxygen concentration profiles made along the vertical axis and the thickness of the tissues estimated from cross-sectional images obtained noninvasively by an optical coherence tomography system. Using our novel system, we obtained (i) oxygen concentration just above the tissues, (ii) gradient of oxygen along vertical axis formed above the tissues within culture medium, and (iii) gradient of oxygen formed within the tissues in real-time. Investigating whether these three parameters could be used to evaluate engineered tissues during culturing, we found that only the third parameter was a good candidate. This implies that the activity of living engineered tissues can be monitored in real-time by measuring the oxygen gradient within the tissues. The proposed measuring strategy can be applied to developing more efficient culturing methods to support the fabrication of engineered thick tissues, as well as providing methods to confirm the quality in real-time.

Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge

Confluent cultured cells on a temperature‐responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three‐dimensional (3‐D) tissue can be fabricated by the layering of cell sheets. A resulting 3‐D multilayered cell sheet‐tissue on a temperature‐responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3‐D multilayered constructs, we attempted to layer cell sheets on a temperature‐responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22‐23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36‐37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet‐layering process. The heating centrifugation shortened the fabrication time by one‐fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue‐fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy.