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.

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.

Optimal flip angle of Gd-EOB-DTPA-enhanced MRI in patients with hepatocellular carcinoma and liver metastasis.

PURPOSE: To investigate optimal flip angle (FA) of three-dimensional fat-suppressed T1-weighted image on Gd-EOB-DTPA-enhanced MRI. METHODS: Forty-five patients with 35 hepatocellular carcinomas (HCCs) and 16 liver metastases (METs) were investigated. Signal-to-noise ratio (SNR), tumor-to-liver contrast (TLC) of HCC and MET, visual image quality (IQ) and lesion conspicuity (LeCo) were evaluated at hepatobiliary phase with different FAs (FA15°-30°-45°-60° in 13 patients, FA5°-10°-15°-20°-25° in 32 patients). RESULTS: TLC gradually showed better in range from FA15° to FA60° and FA5° to FA25°, but SNRs gradually decreased. SNR and TLC-MET at FA15° were significantly better than those at FA45° and FA60°. SNR at FA10° was significantly higher than at FA5°, FA20°, and FA25°. TLC-HCC and TLC-MET at FA5° were inferior to other FAs. IQs and LeCos at FA15° and FA30° were superior to those at FA45° and FA60°. IQs at FA5° and FA25° were significantly lower than those at FA10°-20°, although LeCos for HCC and MET at FA25° were superior to those at FA5°-20°. CONCLUSIONS: FA ranging from 10° to 20° is suitable for hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI, to image HCC and MET.

Hypovascular nodules in patients with chronic liver disease: risk factors for development of hypervascular hepatocellular carcinoma.

PURPOSE: To identify patient characteristics and magnetic resonance (MR) imaging findings associated with subsequent hypervascularization in hypovascular nodules that show hypointensity on hepatobiliary phase gadoxetic acid-enhanced MR images in patients with chronic liver diseases. MATERIALS AND METHODS: Institutional review board approval was obtained, and informed consent was waived. At multiple follow-up gadoxetic acid-enhanced MR imaging examinations of 68 patients, 160 hypovascular nodules were retrospectively reviewed. A Cox regression model for hypervascularization was developed to explore the association of baseline characteristics, including patient factors (Child-Pugh classification, etiology of liver disease, history of local therapy for hepatocellular carcinoma [HCC], and coexistence of hypervascular HCC) and MR imaging findings (fat content, signal intensity on T2-weighted images, and nodule size). In addition, the growth rate was calculated as the reciprocal of tumor volume doubling time to investigate its relationship with subsequent hypervascularization by using receiver operating characteristic and Kaplan-Meier analyses. RESULTS: The prevalence of subsequent hypervascularization was 31% (50 of 160 nodules). Independent Cox multivariable predictors of increased risk of hypervascularization were hyperintensity on T2-weighted images (hazard ratio [HR] = 8.7; 95% confidence interval [CI]: 3.6, 20.8), previous local therapy for hypervascular HCC (HR = 5.0; 95% CI: 1.8, 13.6), Child-Pugh B cirrhosis (HR = 3.6; 95% CI: 1.4, 9.5) and coexistence of hypervascular HCC (HR = 2.0; 95% CI: 1.0, 3.8). The mean growth rate was significantly higher in nodules that showed subsequent hypervascularization than in those without hypervascularization. Kaplan-Meier analysis based on the receiver operating characteristic cutoff level (1.8 × 10(-3)/day [tumor volume doubling time, 542 days]) showed that nodules with a higher growth rate had a significantly higher incidence of hypervascularization (P = 5.2 × 10(-8), log-rank test). CONCLUSION: Hyperintensity on T2-weighted images is an independent and strong risk factor at baseline for subsequent hypervascularization in hypovascular nodules in patients with chronic liver disease. Tumor volume doubling time of less than 542 days was associated with a high rate of subsequent hypervascularization.

Endometrial cancer: preoperative staging using three-dimensional T2-weighted turbo spin-echo and diffusion-weighted MR imaging at 3.0 T: a prospective comparative study.

OBJECTIVES: To prospectively assess the efficacy of 3-T magnetic resonance (MR) imaging using the three-dimensional turbo spin-echo T2-weighted and diffusion-weighted technique (3D-TSE/DW) compared with that of conventional imaging using the two-dimensional turbo spin-echo T2-weighted and dynamic contrast-enhanced technique (2D-TSE/DCE) for the preoperative staging of endometrial cancer, with pathological analysis as the reference standard. METHODS: Seventy-one women with endometrial cancer underwent MR imaging using 3D-TSE/DW (b = 1,000 s/mm(2)) and 2D-TSE/DCE. Two radiologists independently assessed the two imaging sets. Accuracy, sensitivity, and specificity for staging were analysed with the McNemar test; the areas under the receiver operating characteristic curve (Az) were compared with a univariate z-score test. RESULTS: The results for assessing deep myometrial invasion, accuracy, sensitivity, specificity and Az, respectively, were as follows: 3D-TSE/DW-observer 1, 87 %, 95 %, 85 % and 0.96; observer 2, 92 %, 84 %, 94 % and 0.95; 2D-TSE/DCE-observer 1, 80 %, 79 %, 81 % and 0.89; observer 2, 86 %, 84 %, 87 % and 0.86. Most of the values were higher with 3D-TSE/DW without significant differences (P > 0.12). For assessing cervical stromal invasion, there were no significant differences in those values for both observers (P > 0.6). CONCLUSIONS: Accuracy of 3D-TSE/DW was at least equivalent to that of the conventional technique for the preoperative assessment of endometrial cancer. KEY POINTS: • New techniques in MR imaging help assess patients with endometrial cancer. • A 3D T2-weighted TSE sequence seems equally as accurate as conventional techniques. • Three-dimensional TSE/DW imaging does not require intravenous contrast material and is relatively quick. • Tumour extent of endometrial cancer can be clearly shown on diffusion-weighted images. • Junctional zone can be visualised well on 3D-TSE T2-weighted images.

Optimal scan timing of hepatic arterial-phase imaging of hypervascular hepatocellular carcinoma determined by multiphasic fast CT imaging technique.

BACKGROUND: A new multiphasic fast imaging technique, known as volume helical shuttle technique, is a breakthrough for liver imaging that offers new clinical opportunities in dynamic blood flow studies. This technique enables virtually real-time hemodynamics assessment by shuttling the patient cradle back and forth during serial scanning. PURPOSE: To determine optimal scan timing of hepatic arterial-phase imaging for detecting hypervascular hepatocellular carcinoma (HCC) with maximum tumor-to-liver contrast by volume helical shuttle technique. MATERIAL AND METHODS: One hundred and one hypervascular HCCs in 50 patients were prospectively studied by 64-channel multidetector-row computed tomography (MDCT) with multiphasic fast imaging technique. Contrast medium containing 600 mg iodine per kg body weight was intravenously injected for 30 s. Six seconds after the contrast arrival in the abdominal aorta detected with bolus tracking, serial 12-phase imaging of the whole liver was performed during 24-s breath-holding with multiphasic fast imaging technique during arterial phase. By placing regions of interest in the abdominal aorta, portal vein, liver parenchyma, and hypervascular HCCs on the multiphase images, time-density curves of anatomical regions and HCCs were composed. Timing of maximum tumor-to-liver contrast after the contrast arrival in the abdominal aorta was determined. RESULTS: For the detection of hypervascular HCC at arterial phase, mean time and value of maximum tumor-to-liver contrast after the contrast arrival were 21 s and 38.0 HU, respectively. CONCLUSION: Optimal delay time for the hepatic arterial-phase imaging maximizing the contrast enhancement of hypervascular HCCs was 21 s after arrival of contrast medium in the abdominal aorta.

Importance of beating rate control for the analysis of drug effects on contractility in human induced pluripotent stem cell-derived cardiomyocytes.

Cardiac contractility evaluation using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has recently attracted much attention as a clinical cardiotoxicity predictive model. Most studies on this were conducted under spontaneous beating conditions and involved video-based analyses. Cardiac contractility is known to be influenced by beating rates; accordingly, beating rate control is recommended to accurately analyze the effects of drugs on cardiac contractility. Therefore, we investigated the relationship between contraction parameters and beating rates of cardiac cell sheet tissues by directly measuring the contraction force and compared the effects of ion channel drugs (mexiletine, ranolazine, and dofetilide) on contraction parameters under spontaneous beating conditions with those under pacing (1 Hz) conditions. To characterize the contraction/relaxation kinetics, we introduced a novel analysis tool, called a "C-V loop," a plot of contraction force versus force-changing rate ("velocity"). When we increased the beating rate, the contraction force, force-changing rate, and relaxation time markedly decreased. The occurrence frequencies of beating arrest and irregular beats at high concentration ranges of mexiletine and ranolazine were more suppressed in paced samples than in spontaneously beating ones. We also found that relaxation time increased by treatment with dofetilide and contraction amplitude decreased in a concentration-dependent manner by mexiletine treatment only in the samples under pacing. These drug responses were consistent with the previous reports using human samples. These results indicated that beating rate control is necessary to stably evaluate the effects of drugs on contractility and that tests under 1-Hz pacing are more relevant to clinical settings.

Optimal scanning protocol of arterial dominant phase for hypervascular hepatocellular carcinoma with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced MR.

PURPOSE: To investigate optimal delay time of hepatic arterial phase in Gadoxetate-enhanced MR for detecting hypervascular hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Forty-five patients with 85 hypervascular HCCs and 9 patients with 16 hypervascular HCCs underwent Gadoxetate- and Gd-DTPA-enhanced MR at 1.5 Tesla (T) system, respectively. All HCCs were analyzed 10-38 s after injection using a time-resolved dynamic MR sequence with keyhole data sampling. Seven sequential phase images (1 phase = 4 s) were obtained during a single breath hold of 28 s. Time-intensity curves of the abdominal aorta, liver parenchyma, and HCC were obtained, then aortic contrast arrival time, time of peak HCC enhancement, duration time of HCC and aortic enhancement, and time delay from aortic contrast arrival to peak enhancement of HCC were measured. RESULTS: Aortic contrast arrival time was 15.1 ± 2.9 s, time of peak HCC enhancement 29.9 ± 4.6 s, duration time of HCC enhancement 17.4 ± 6.4 s postinjection of Gadoxetate. Duration of aortic enhancement (23.6 ± 3.5 s) of Gadoxetate-enhanced MR was significantly less than that of Gd-DTPA-enhanced MR (26.3 ± 2.8 s) (P < 0.0059). CONCLUSION: Peak enhancement time of HCC on Gadoxetate-enhanced MR imaging occurred at 14.6 ± 4.6 s after aortic contrast arrival.

Unenhanced fat fraction ratios obtained by MR and enhanced T2* values with liver-specific MR contrast agents for diagnosis of non-alcoholic steatohepatitis in rats.

BACKGROUND: Non-invasive MR imaging is expected to be used for accurate diagnosis and quantification of non-alcoholic steatohepatitis (NASH), because NASH is a progressive fatty liver disease. New MR techniques, such as fat fraction ratio (FFR) and T2* value measurement, have attracted an increasing attention, because those techniques can measure quantitative parameters of fibrosis, fat and iron deposition in the liver. PURPOSE: To investigate the potential of FFR and T2* value in NASH with pre-enhancement, gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) or super-paramagnetic iron oxide (SPIO)-enhanced MRI. MATERIAL AND METHODS: Twenty-eight rats were divided equally into four groups (one control group and three NASH groups). All rats underwent unenhanced, Gd-EOB-DTPA, and SPIO-enhanced MRI. The T2* value of the liver was measured for each image sequence, and then changes in T2* values before and after each injection were analyzed using Dunnett's test. The reduction rate of T2* value before and 13 min after injection of Gd-EOB-DTPA or SPIO was analyzed using Mann-Whitney's U test. Moreover, FFR of the liver was measured before enhancement, and the relationship between fat fraction and the calculated fat area percentage on a pathological specimen was examined using Spearman's correlation test. RESULTS: On pre-enhancement, FFR and T2* value were 26.0% ± 12.0% and 21.5 ± 4.2 ms for all NASH groups, and 0.9% ± 0.5% and 30.8 ±-5.5 ms for control, respectively. Both FFR and T2* values were significantly different between the NASH and control groups. The reduction rate of T2* value was significantly lower in the NASH groups than in the control group on SPIO-enhanced MRI, though there was no significant difference on Gd-EOB-DTPA-enhanced MRI. FFR was correlated with the calculated fat area percentage for the pathological specimen. CONCLUSION: Pre-enhancement FFR, T2* value measurement and reduction rate of T2* value on SPIO-enhanced MRI may help estimate the progress of liver fat deposition and fibrosis in NASH.

Functional Analysis of Induced Human Ballooned Hepatocytes in a Cell Sheet-Based Three Dimensional Model.

BACKGROUND: Ballooned hepatocytes (BH) are a key histological hallmark of nonalcoholic steatohepatitis (NASH), yet their consequences for liver-specific functions are unknown. METHODS: In our previous study, an experimental model of human induced-BHs (iBH) has been successfully developed based on cell sheet technology. This study aimed to determine the functions of iBHs in the primary human hepatocyte/normal human dermal fibroblast (PHH/NHDF) co-culture cell sheets. Normal hepatocytes in the PHH/3T3-J2 co-culture cell sheets were set as a control, since 3T3-J2 murine embryonic fibroblasts have exhibited previously long term maintenance of PHH functions. RESULTS: It was found that, albumin secretion was not affected in iBHs, but urea synthesis as well as cytochrome P450 enzyme (CYP) activities including CYP1A2 and CYP3A4, were significantly reduced in iBHs. Besides, loss of bile canaliculi was observed in iBHs. These findings are consistent with clinical studies of human NASH. In addition, PHH/NHDF cell sheets demonstrated two fold higher TGF-ß1 secretion compared with PHH/3T3-J2 cell sheets. Furthermore, treatment with a TGF-ß inhibitor and a semi-synthetic bile acid analogue (obeticholic acid, phase 3 trial of NASH therapy) ameliorated the histological appearance of established iBHs. CONCLUSION: In summary, this study demonstrates the priority of iBHs in recapitulating not only histology but also clinically relevant hepatic dysfunctions in human NASH and suggests TGF-ß and bile acid related signal pathway may play important roles in the formation of iBHs.

Garlic oil suppresses high-fat diet induced obesity in rats through the upregulation of UCP-1 and the enhancement of energy expenditure.

Garlic (Allium sativum L.) has long been used as a medicinal food. Indeed, garlic and its constituents have been shown to possess potent regulatory activities in bodily functions, including blood coagulation, lipid metabolism, immunity and xenobiotic metabolism. In this study, we aimed to examine the anti-obesity effects of garlic oil and to elucidate the possible underlying mechanisms. For this purpose, garlic oil (GO; 80 mg/kg body weight, p.o.) or corn oil alone as a vehicle-control were administered to male Sprague-Dawley rats every other day for 10 weeks. The results revealed that GO administration significantly reduced body weight gain and white adipose tissue (WAT) mass, which had been increased by feeding on the AIN-76-based high-fat diet (60% kcal fat). Expired gas analysis was performed at 9 weeks following the GO administration to calculate fuel oxidation. GO administration enhanced O2 consumption during the dark period (at night) and increased energy expenditure through fat oxidation during the light period (daytime); however, carbohydrate oxidation remained unaltered. Western blot analysis revealed that GO administration increased UCP1 protein expression in brown adipose tissue (BAT). On the whole, the findings of this study indicated that GO suppressed body weight gain and WAT mass in the rat model of high-fat diet-induced obesity by increasing UCP1 expression and by enhancing fat oxidation and energy expenditure.

In Vitro Production of Human Ballooned Hepatocytes in a Cell Sheet-based Three-dimensional Model.

Ballooned hepatocytes (BH) are enlarged, abnormal hepatocytes, which are usually involved in liver diseases, in particular, nonalcoholic steatohepatitis (NASH). However, formation of BHs in vitro has been seldom reported. This study reported an in vitro strategy to produce human BHs in a cell sheet-based three-dimensional (3D) model where primary human hepatocytes were cocultured with normal human dermal fibroblasts. Enlargement of hepatocytes (2.3 times larger than normal, p < 0.01), loss of cytoplasmic keratin, appearance of Mallory-Denk bodies (MDBs), and abundant fat droplets accumulation were observed after only a few days culture. Additionally, ultrastructural characteristic findings of BHs in human NASH, including enlarged mitochondria with crystalline inclusions, dilated endoplasmic reticulum, and MDBs formation were also observed in the 3D model. Furthermore, pathophysiological features of human NASH, such as increased secretion of sonic hedgehog ligands and myofibroblast activation were found. This study reports in vitro production of human BHs by using a cell sheet-based 3D model. Similar histological, ultrastructural, and pathophysiological features to human NASH are discovered in this model. This model may facilitate study of BHs and increase our knowledge of the pathogenesis of human liver diseases. Impact Statement Human ballooned hepatocytes (BH), which are present in nonalcoholic steatohepatitis (NASH) are mainly studied based on human liver biopsies and animal models. In this study, human BHs can be successfully reproduced in a cell sheet-based in vitro model, which, as far as we know, is the first in vitro model that recapitulates so many histological and ultrastructural hallmarks of BHs found in human NASH. Additionally, this study also demonstrated presence of some NASH pathophysiological features. This model may facilitate the study of hepatocellular ballooning and prove beneficial in translational preclinical drug discovery in NASH.

Synchronization and spatiotemporal patterns in coupled phase oscillators on a weighted planar network.

To reveal the relation between network structures found in two-dimensional biological systems, such as protoplasmic tube networks in the plasmodium of true slime mold, and spatiotemporal oscillation patterns emerged on the networks, we constructed coupled phase oscillators on weighted planar networks and investigated their dynamics. Results showed that the distribution of edge weights in the networks strongly affects (i) the propensity for global synchronization and (ii) emerging ratios of oscillation patterns, such as traveling and concentric waves, even if the total weight is fixed. In-phase locking, traveling wave, and concentric wave patterns were, respectively, observed most frequently in uniformly weighted, center weighted treelike, and periphery weighted ring-shaped networks. Controlling the global spatiotemporal patterns with the weight distribution given by the local weighting (coupling) rules might be useful in biological network systems including the plasmodial networks and neural networks in the brain.

Adrenal masses: the value of additional fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) in differentiating between benign and malignant lesions.

OBJECTIVE: To investigate whether integrated fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) can differentiate benign from adrenal malignant lesions on the basis of maximum standardized uptake value (SUV(max)), tumor/liver (T/L) SUV(max) ratio, and CT attenuation value (Hounsfield Units; HU) of unenhanced CT obtained from FDG-PET/CT data. METHODS: We studied 30 patients with 35 adrenal lesions (16 adrenal benign lesions, size 16 +/- 5 mm, in 15 patients; and 19 adrenal malignant lesions, 24 +/- 12 mm, in 15 patients) who had confirmed primary malignancies (lung cancer in 23 patients, lymphoma in 2, esophageal cancer in 2, hypopharyngeal cancer in 1, prostate cancer in 1, and 1 patient in whom lesions were detected at cancer screening). All patients underwent PET/CT at 1 h post FDG injection. Diagnosis of adrenal malignant lesions was based on interval growth or reduction after chemotherapy. An adrenal mass that remained unchanged for over 1 year was the standard used to diagnose adrenal benign lesions. Values of FDG uptake and CT attenuation were measured by placing volumetric regions of interest over PET/CT images. Adrenal uptake of SUV(max) >/= 2.5 was considered to indicate a malignant lesion; SUV(max) < 2.5 was considered to indicate a benign lesion. In further analysis, 1.8 was employed as the threshold for the T/L SUV(max) ratio. Unenhanced CT obtained from PET/CT data was considered positive for adrenal malignant lesions based on a CT attenuation value >/= 10 HU; lesions with a value < 10 HU were considered adrenal benign lesions. Mann-Whitney's U test was used for statistical analyses. RESULTS: SUV(max) in adrenal malignant lesions (7.4 +/- 3.5) was higher than that in adrenal benign lesions (2.1 +/- 0.5, p < 0.05). The CT attenuation value of adrenal malignant lesions (27.6 +/- 11.9 HU) was higher than that of adrenal benign lesions (10.1 +/- 12.3 HU, p < 0.05). In differentiating between adrenal benign and malignant lesions, a CT threshold of 10 HU corresponded to a sensitivity of 57%, specificity of 94%, accuracy of 74%, positive predictive value of 92% and negative predictive value of 65%. An SUV(max) cut-off value of 2.5 corresponded to a sensitivity of 89%, specificity of 94%, accuracy of 91%, positive predictive value of 94% and negative predictive value of 88%. The T/L SUV(max) ratio was 1.0 +/- 0.2 for adrenal benign lesions and 4.5 +/- 3.0 for adrenal malignant lesions. And T/L SUV(max) ratio cut-off value of 1.8 corresponded to a sensitivity of 85%, specificity of 100%, accuracy of 91%, positive predictive value of 100% and negative predictive value of 83%. CONCLUSIONS: FDG-PET/CT with additional SUV(max) analysis improves the diagnostic accuracy of adrenal lesions in cancer patients.

Analysis of force vector field during centrifugation for optimizing cell sheet adhesion.

A three-dimensional tissue was fabricated by layering cell sheets with centrifugation. In this system, an optimal centrifugal force promoted the adhesion between (a) a cell sheet and a culture dish, and (b) layered cell sheets, resulting in a significant decrease in the fabrication time of the tissue. However, negative effects like sliding/significant deformation of cell sheets were observed upon high rotational speed use. These negative effects inhibit the further shortening of the fabrication time. The sliding/deformation suggests that the centrifugal forces were applied on the cell sheets in unwanted directions. Studies on the force vector field applied to the object placed on the plate during centrifugation are not available, and thus, the reason for the occurrence of such negative effects is unclear. Here, we theoretically derived the spatial distribution of acceleration applied on a plate during centrifugation. Using this theory, we found that the negative effects were triggered by the centrifugal force in the direction parallel to the plate surface, which appeared due to an inclination of the plate surface against a horizontal plane. Therefore, by adding weights on the plate edge to maintain the plate surface in a horizontal position, we succeeded in eliminating the negative effects and in increasing the rotational speed, with the minimum risk of sliding/deformation of cell sheets. We succeeded in reducing the time to establish tight adhesion between a mouse myoblast sheet and a culture dish, and layered cell sheets by increasing the centrifugal force from 5 min to 1 min without significant cytotoxicity.

Rapid creation system of morphologically and functionally communicative three-dimensional cell-dense tissue by centrifugation.

This study reports a rapid fabrication system of a morphologically and functionally communicative three-dimensional (3D) cell-dense tissue without scaffolds by centrifugation. The tight adhesion between C2C12 myoblasts and culture surface was accelerated without significant cell damage by centrifugation (80 x g, 37 °C, 30 min). A thicker tissue created on a temperature-responsive culture surface was harvested by decreasing temperature. The 3D myoblast tissues having approximately 200 µm-thickness were created at 1.5 h [centrifugation (80 x g, 37 °C) for 30 min and tissue harvest for 1 h]. However, in the case of without centrifugation, the myoblast tissues had fragile parts even at 7.5 h after the incubation. Additionally, electrically/functionally communicative and thicker human induced pluripotent stem (iPS) cell-derived cardiac tissues were created rapidly by the centrifugation and cultivation at 37 °C. We report a centrifugation system that significantly shortens the creation time of 3D tissues. We envision that this procedure will contribute to the field of tissue engineering and regenerative medicine. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1447-1453, 2018.

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. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:692-701, 2018.

Low-temperature culturing improves survival rate of tissue-engineered cardiac cell sheets.

Assembling three-dimensional (3D) tissues from single cells necessitates the use of various advanced technological methods because higher-density tissues require numerous complex capillary structures to supply sufficient oxygen and nutrients. Accordingly, creating healthy culture conditions to support 3D cardiac tissues requires an appropriate balance between the supplied nutrients and cell metabolism. The objective of this study was to develop a simple and efficient method for low-temperature cultivation (< 37 °C) that decreases cell metabolism for facilitating the buildup of 3D cardiac tissues. We created 3D cardiac tissues using cell sheet technology and analyzed the viability of the cardiac cells in low-temperature environments. To determine a method that would allow thicker 3D tissues to survive, we investigated the cardiac tissue viability under low-temperature culture processes at 20-33.5 °C and compared it with the viability under the standard culture process at 37 °C. Our results indicated that the standard culture process at 37 °C was unable to support higher-density myocardial tissue; however, low-temperature culture conditions maintained dense myocardial tissue and prevascularization. To investigate the efficiency of transplantation, layered cell sheets produced by the low-temperature culture process were also transplanted under the skin of nude rats. Cardiac tissue cultured at 30 °C developed denser prevascular networks than the tissue cultured at the standard temperature. Our novel findings indicate that the low-temperature process is effective for fabricating 3D tissues from high-functioning cells such as heart cells. This method should make major contributions to future clinical applications and to the field of organ engineering.

Effect of fluctuation in step size on actin-myosin sliding motion.

It is possible that the step size, or power stroke, of a skeletal muscle myosin is not constant; rather, it fluctuates for each force generation. The estimated widths of the fluctuation are as large as the estimated values of the step size. Although such non-negligible fluctuation is presumed to affect the sliding motion, these effects remain unclear. We examined a system driven by a single myosin molecule sliding along an actin filament to reveal its basic effects. First, we calculated the sliding velocity of the system for each fluctuation width and found that the mean velocity increased with the fluctuation width. We also found that the estimated fluctuation widths satisfied the conditions for maximizing the sliding velocity. Next, we examined the sliding motion along a heterogeneous filament, on which binding sites for myosins were distributed randomly. We found that the loss in sliding velocity that was attributable to heterogeneity of the filament became small when fluctuation in the step size existed. This finding implied that the fluctuation stabilized velocity sliding along possible heterogeneous filaments. These benefits of fluctuation in step size might be used in biological systems, such as a muscle system, and are applicable to fabricated micromachines.

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. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 855-864, 2017.