Microfluidic devices are powerful bioapplication tools for cellular experiments in particular, as they can regulate physical and chemical parameters such as the flow rate, shear stress, oxygen, and ...molecular concentrations in a culture medium to mimic physiological and pathological microenvironments in vitro. However, as cell cultures take place in enclosed spaces, culture samples have to be removed repeatedly to monitor cell-derived metabolites and proteins in order to maintain the cellular microenvironment. We report a simple method for obtaining surface enhanced Raman scattering (SERS) spectra through self-assembly of a nanoparticle monolayer on polydimethylsiloxane (PDMS), which is commonly used in highly biocompatible microfluidic devices. Silica nanoparticles were stabilized as a hexagonal close packed structure on an O2 plasma-processed PDMS membrane, and was coated with silver using vapor deposition to create an SERS plate. When this SERS plate was installed in a microfluidic device, the nanoparticles did not peel off even after long-term fluid immersion. The SERS spectra exhibited stable SERS generation and an enhancement factor of more than 1.5 × 106 of the Raman signals from rhodamine 6G compared to the signals without nanoparticles. The SERS spectra of lactate and ATP were obtained, and Raman shifts due to the different masses of 12C- and 13C-lactate were observed, suggesting that the proposed method can be applied to determine the cellular metabolic flux in microfluidic devices. We also obtained cell membrane-derived SERS spectra by culturing murine mammary carcinoma 4T1 cells directly on the nanoparticle membrane. PDMS has high biocompatibility and is often used for fabricating microfluidic devices. Through tight binding of the nanoparticles to the O2 plasma-treated PDMS, the chemical reaction products or the metabolites from cells can be measured for a long duration, while maintaining the microenvironment. We anticipate that this technology can be utilized as a useful tool for analyzing cellular metabolic functions and imaging cellular distributions without staining in various pathological models created in microfluidic devices.
Hypoxia-related mechanisms are important in tumor biology and immune responses. Oxygen is delivered to tumor tissue by blood flowing through abnormal and dysfunctional microvessels, resulting in ...heterogeneity of tissue oxygenation within the tumor. Hypoxic conditions play a role in directing angiogenesis, guiding immune cells, and inducing tumor metastasis. Mimicking such oxygen gradient in in vitro cellular experiments is important to clarify the mechanisms involved in tumor biology. Previous research has led to the development of cell culture devices that generate an oxygen gradient, but it was impossible to monitor the oxygen gradient during cell culture. In this study, we designed an open-well polydimethylsiloxane (PDMS) microfluidic device integrated with an oxygen-sensitive film, which permits oxygen measurement around cells and molecular analysis after cell culture experiments. Mathematical simulation and phosphorescence-based partial oxygen measurements show that the gradient can be controlled by changing the oxygen gas concentration inside the microchannels, according to the requirements of various biological models. A monoculture of endothelial cells exposed to an oxygen gradient in the device showed increased expression of oxygen-responsive genes in the hypoxic area. These results suggest that our microfluidic device can be used for in vitro experiments such as gene expression and migration assays. We believe that this new device is a powerful tool for studies of tumor biology and immunology.
Immature neovessels in tumors lead to the formation of heterogeneous hypoxic areas within tumors. Such hypoxic conditions not only reduce the effectiveness of radiation treatment but are also related ...to tumor invasion and metastasis. To better understand tumor-related mechanisms, it is important to quantitate the temporal and spatial changes of tumor hypoxia. However, no useful technique capable of measuring oxygen concentration in vivo has been proposed to date. We aimed to develop a microscope system to measure oxygen metabolism in tumor tissues using a laser-assisted oxygen quenching method. A scanning laser with galvano mirrors was capable of imaging tissue hypoxia and allowed configuration of time and spatial resolution by altering the laser spot size, scan rate, or scan distance. We examined the feasibility of the system by in vitro oxygen measurement, and applied this method to in vivo imaging of tumor oxygenation during oxygen inhalation in tumor-implanted mice. Oxygen tension inside tumors increased soon after oxygen inhalation, but decreased gradually after 20 min in spite of continuous oxygen inhalation, indicating that this model replicates the clinical experience that long-term tumor oxygenation cannot be achieved by oxygen inhalation. Quantitative analysis of tumor oxygenation may help reveal the mechanisms of oxygen metabolism in tumor tissues, leading to the development of more effective radiation therapy.
•Microdevice with oxygen gradient can mimic hepatic microenvironment in vitro.•pO2 on culture area and cellular O2 consumption can be quantified during culture.•PEPCK and GK expressions in primary ...hepatocytes changed depending on pO2.•Microdevice can generate various O2 gradients by changing micro gas-channels.
In a hepatic lobule, different sets of metabolic enzymes are expressed in the periportal (PP) and pericentral (PC) regions, forming a functional zonation, and the oxygen gradient is considered a determinant of zone formation. It is desirable to reproduce lobular microenvironment in vitro, but incubation of primary hepatocytes in conventional culture dishes has been limited at fixed oxygen concentrations due to technical difficulties.
We designed a cell culture microdevice with an oxygen gradient to reproduce the hepatic microenvironment in vitro. The oxygen gradient during cell culture was monitored using a laser-assisted phosphorescence quenching method, and the cellular oxygen consumption rate could be estimated from changes in the gradient. Culture medium was continuously exchanged through microchannels installed in the device to maintain the oxygen gradient for a long term without transient hyper-oxygenation.
The oxygen consumption rates of hepatocytes at 70.0mmHg and 31.4mmHg of partial oxygen pressure, which correspond to PP and PC regions in the microdevice, were 3.67×10−10 and 3.15×10−10mol/s/106 cells, respectively. Antimycin A changed the oxygen gradient profile, indicating that cellular respiration can be estimated during cell culture. RT-PCR analysis of hepatocytes cultured under the oxygen gradient showed that mRNA expression of PEPCK and GK significantly increased in culture areas corresponding to PP and PC regions, respectively.
These results indicate that the developed microdevice can reproduce the hepatic lobular microenvironment. The oxygen gradient in the microdevice can be closely controlled by changing the sizes of gas channels and the ambient oxygen concentration around the device; therefore, it could be expected to mimic the oxygen gradient of various organs, and it may be applicable to other pathological models.
Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the ...near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology.
Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light.
Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature.
Results: A specific combination of wavelengths at low irradiances (250 to 400 mW / cm2 for 1064 nm and 55 to 65 mW / cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes.
Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.
Goal: For personalized clinical applications, flexible conductors require both high electrical conductivity and resistance to stretching and bending. Here, we developed a two-dimensional array ...sinusoidal wave (TDAS) conductor, characterized its electrical properties under stretching and bending loads, and measured photoelectric pulse waves. Methods: TDAS structures with wavelengths of 500−2000 μm and amplitudes of 50−200 μm were microfabricated on Al substrates. These structures were then transferred to dimethylpolysiloxane, followed by Au sputtering to obtain TDAS conductors. Results: TDAS conductors with a 200-μm amplitude suppressed the increase in resistance to stretching and bending and maintained conductivity >30% stretching. The small cracks in the valleys observed with electron microscopy contributed to its stretching properties. The connection of LEDs and photodiodes to the TDAS conductors enabled fingertip pulse wave detection. Conclusions: Film-type TDAS conductors, which can maintain high conductivity during stretching and bending, have potential for stress-free physiological monitoring of organs such as the heart as well as the body surface.
We newly developed high-performance blood coagulation nanofibres using Polycaprolactone (PCL) including Calcium carbonate (CaCO3) and β-chitosan. The most important feature for a wound dressing is ...that it is harmless to the human body. Here, we fabricated the nanofibre using all human-safe materials. We used a PCL nanofibre mat as a substrate and then synthesized artificial CaCO3 from sodium carbonate and calcium chloride. The CaCO3 was then added to the fibre solution to create PCL/CaCO3 nanofibres. We coated PCL and PCL/CaCO3 nanofibres with β-chitosan as a hemostatic material via a spray method. For the more uniform coating, we used ultrasonic spray coating method and then compared the blood coagulation abilities of the PCL and PCL/CaCO3 nanofibres. We found that the PCL/CaCO3 nanofibres sprayed with β-chitosan had a greater effect on blood coagulation than the PCL nanofibres. In the result of animal experiment, β-chitosan have a key role in changing of surface wettability from hydrophobic to hydrophilic. Moreover, this is contributed to enhance blood coagulability. PCL/CaCO3 nanofibres sprayed with β-chitosan therefore offer promise in medical applications.
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•The materials which used to fabricate nanofibre were all human-safety.•β-chitosan had a key role in changing of PCL surface wettability from hydrophobic to hydrophilic.•The presence of CaCO3 was determined blood clotting rate.•PCL/CaCO3 nanofibre sprayed with β-chitosan exhibited highest blood clotting ability.
Activity of blood cells, erythrocytes, leucocytes, and platelets, in microcirculation was observed by using an intravital microscope and confocal laser scanning microscope connected with an image ...processing system including fluorescence and phosphorescence emission methods. Dynamic functions of the blood flow were mainly observed in mesentery, brain, and liver tissues of rats. The results are summarized as follows: Deformability of diabetic erythrocytes was significantly lower than that of healthy controls, particularly at high shear rate. The spring constant and Young’s modulus of diabetic erythrocytes obviously stiffened, making them hard to deform in the capillary. During hemorrhagic shock and thrombosis, flow velocity and oxygen partial pressure of blood decreased in the brain and liver tissues that can be visualized by using FITC stained erythrocytes and Pd-porphyrin derivative as a pO2 probe. Platelet adhesion and thrombus formation in the micro-vessels accelerated under the photodynamic reaction; diabetic platelets showed augmented adhesion and aggregation on the vessel wall which was followed by acute thromboembolism. Active oxygen radicals take part in thrombus formation, accompanied with adhesion of the activated leucocytes. Fluorescent dye probes, rhodamine G and acridine orange, are quite useful for visualization of the flow behavior of platelets and leucocytes, respectively.
1 Department of Physiology, Kawasaki Medical School, Kurashiki, 701-0192; 2 Department of Clinical Research, National Saitama Hospital; Wako, 351-0102; 3 Oshio Clinic, Chiyoda-ku, 101-0063; and 4 ...Department of Applied Physics and Physico-Informatics, Faculty of Science and Technology, Keio University, Yokohama, 223-8522 Japan
Submitted 23 July 2003
; accepted in final form 2 December 2003
Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.
cerebral microcirculation; erythrocyte velocity; oxygen tension; fluorescence; phosphorescence
Address for reprint requests and other correspondence: K. Tsukada, Dept. of Physiology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 Japan (E-mail: ktsukada{at}med.kawasaki-m.ac.jp ).
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