A perfusable microfluidic device with on-chip total internal reflection fluorescence microscopy (TIRFM) for in situ and real-time monitoring of live cells

A microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis was developed. This integrated device enables in situ Total Internal Reflection Fluorescence Microscopy (TIRFM) on adherent cells cultured under continuous medium perfusion. This TIR-based chip, allows TIRFM to be easily performed on cells without the assembly of complicated optical components and cell culture chambers. The integrated device was evaluated by tracking the movement of fluorescent beads and monitoring the location of insulin granules in mouse pancreatic β-cells. This system offers higher signal-to-noise (S/N) ratio than epi-fluorescence microscopy (EPIFM), and comparable image quality to commercial TIRFM systems when imaging insulin granules. We also detected repetitive changes in intracellular Ca(2+) concentration in MIN6-m9 cells stimulated with KCl, which demonstrates quick perfusion for cell analysis while maintaining high S/N ratio.     

CoSTEL: a computer peripheral remote sensing device for 3-dimensional monitoring of human motion

Medical & Biological Engineering & Computing - An automatic system (CoSTEL) which permits the reconstruction of the instantaneous 3-dimensional position of target points (infra-red light...     

Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.     

Multicompartmented microfluidic device for characterization of dose-dependent cadmium cytotoxicity in BALB/3T3 fibroblast cells

This paper describes the development of a miniaturized multicompartmented microfluidic device for high-throughput cell cytotoxicity assays and its applicability to the investigation of cadmium-induced cytotoxicity. A steady gradient of cadmium was generated inside the compartments to study the effects of cadmium ion on BALB/3T3 fibroblast cells in a dose-dependent fashion. The device allowed the performance of multiplexed assays to probe the dosage effect of cadmium, morphological alterations of live cells, regulation of proliferation and viability of cells, determination of reactive oxygen species, mechanisms of cell death, i.e. apoptosis and/or necrosis, and immunocytochemical staining of cells in parallel and/or serially, or on a single population simultaneously. The outcomes of all the microfluidic assays were compared to conventional plates-based cytotoxicity assays. The results indicated that the cells cultured in this device were morphologically healthy with greater than 90% viability. They further suggested that the basic mode of cell death behind cadmium-induced cytotoxicity was apoptosis, which was regulated by intracellular oxidative stress via cytoskeleton disorganization and nuclear condensation. Such microenvironments resemble the in vivo physiological conditions very closely and thus offer a unique platform for more accurate observations of cytotoxicity assays and more precise estimation of the IC₅₀ value in comparison to conventional analytical assays. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A microfluidic platform for 3-dimensional cell culture and cell-based assays

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.     

Development of a small wireless device for perspiration monitoring

A small and wireless device that can capture the temporal pattern of perspiration by a novel structure of water vapor collection combined with reusable desiccant has been developed. The novel device consists of a small cylindrical case with a temperature/relative humidity sensor, battery-driven data logger, and silica gel (desiccant). Water vapor of perspiration was detected by the change in relative humidity and then adsorbed by silica gel, allowing continuous recording of perspiration within a closed and wireless chamber, which has not been previously achieved. By comparative experiments using the commercially-available perspiration monitoring device, the developed device could measure perspiration as efficiently as the conventional one, with a normalized cross coefficient of 0.738 with 6 s delay and the interclass correlation coefficient [ICC(2, 1)] of 0.84. These results imply a good agreement between the conventional and developed devices, and thus suggest the applicability of the developed device for perspiration monitoring.     

Dynamic monitoring of single cell lysis in an impedance-based microfluidic device

A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.     

Preparation of monodispersed chitosan microspheres and in situ encapsulation of BSA in a co-axial microfluidic device

This work describes a novel microfluidic method to prepare monodispersed chitosan microspheres by using the solvent extraction method. Our strategy is that a chitosan/acetic acid aqueous solution is emulsified in an organic phase containing the extractant by using the co-flowing shear method in a co-axial microfluidic device. The formed droplets are in situ solidified within a synthesizing channel by the extraction of acetic acid from the chitosan aqueous droplets to the organic solution. Based on this approach, the size of chitosan microspheres can be successfully controlled from 100 μm to 700 μm in diameter with a variation of less than 4%. Furthermore, high loading efficiency (>95%) of Bovine serum albumin (BSA) can be in situ encapsulated. The present method has the advantages of actively controlling the droplet diameter, narrow size distribution, good sphericity, and having a simple and low cost process, with a high throughput. This approach for the preparation of chitosan microspheres will provide many potential applications for pharmaceutical area.     

A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system

We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 μL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.     

A microfluidic device for continuous manipulation of biological cells using dielectrophoresis

The present study demonstrates the design, simulation, fabrication and testing of a label-free continuous manipulation and separation micro-device of particles/biological cells suspended on medium based on conventional dielectrophoresis. The current dielectrophoretic device uses three planner electrodes to generate non-uniform electric field and induces both p-DEP and n-DEP force simultaneously depending on the dielectric properties of the particles and thus influencing at least two types of particles at a time. Numerical simulations were performed to predict the distribution of non-uniform electric field, DEP force and particle trajectories. The device is fabricated utilizing the advantage of bonding between PDMS and SU8 polymer. The p-DEP particles move away from the center of the streamline, while the n-DEP particles will follow the central streamline along the channel length. Dielectrophoretic effects were initially tested using polystyrene beads followed by manipulation of HeLa cells. In the experiment, it was observed that polystyrene beads in DI water always response as n-DEP up to 1 MHz frequency, whereas HeLa cells in PBS medium response as n-DEP up to 400 kHz frequency and then it experiences p-DEP up to 1 MHz. Further, the microscopic observations of DEP responses of HeLa cells were verified by performing trapping experiment at static condition.     

Transfection efficiency for size-separated cells synchronized in cell cycle by microfluidic device

Non-viral system generally demonstrates less efficacious in transgene delivery than viral system; however it represents a safer alternative to viral system. In this study, transfection efficiency for human hepatocellular liver carcinoma cells synchronized in cell cycle at G0/G1 phase, which was sorted in size with a microfluidic device based on hydrodynamic filtration, was investigated by using a reverse transfection method. The synchronized cells were recovered at the yield of 80% from the micro-channel, and green fluorescent protein gene encoding plasmid mixed with lipofectoamine was transfected. The transfection efficiency of the cells at G0/G1 phase was 1.8 times higher than non-synchronized cells. The manipulation of cell cycle status could increase transfection efficiency in non-viral system, indicating size-based cell cycle synchronization is a powerful tool as a noninvasive method for bioscience and biotechnology.     

Sperm motion in a microfluidic fertilization device

Microfluidics has shown promise as a new platform for assisted reproduction. To assess the potential of microfluidics for fertilization, we studied sperm and fluid motion in microchannels to better understand the flow characteristics in a microfluidic device, how sperm interacted with this flow, and how sperm-oocyte attachment occurs in the device. There is a threshold fluid velocity where sperm transition from traveling with the fluid to a regime in which the sperm can move independently of the flow. A significant population of sperm remained in the inlet well area. Based on the lack of progressive forward movement, it was presumed that these sperm may have defects. Also of extreme interest was the tendency of sperm to travel along surface contours. These observations provide an improved understanding of sperm motion in microchannels and provide a basis for improved device designs that take advantage of the sperm/flow and sperm/geometry interactions.     

A novel microbead-based microfluidic device for rapid bacterial identification and antibiotic susceptibility testing

Effective treatment of infectious diseases depends on the ability to rapidly identify the infecting bacteria and the use of sensitive antibiotics. The currently used identification assays usually take more than 72 h to perform and have a low sensitivity. Herein, we present a microbead-based microfluidic platform that is highly sensitive and rapid for bacterial detection and antibiotic sensitivity testing. The platform includes four units, one of which is used for bacterial identification and the other three are used for susceptibility testing. Our results showed that Escherichia coli O157 at a cell density range of 10¹–10⁵ CFU/μL could be detected within 30 min. Additionally, the effects of three antibiotics on E. coli O157 were evaluated within 4–8 h. Overall, this integrated microbead-based microdevice provides a sensitive, rapid, reliable, and highly effective platform for the identification of bacteria, as well as antibiotic sensitivity testing.     

活体荧光成像监测HCV NS3/4A蛋白在小鼠体内的瞬时表达

目的建立HCV NS3/4A蛋白酶在小鼠体内可视化表达模型。方法利用水动力转染技术将融合基因NS3/4A-Fluc转染至小鼠肝脏,建立目的基因的瞬时表达模型,通过RT-PCR方法检测目的基因Fluc及NS3/4A的RNA表达水平,以及通过Western blot方法检测目的基因NS3/4A-Fluc的蛋白表达水平。结果建立了通过报告基因Fluc反映HCV NS3/4A蛋白酶的瞬时表达可视化小鼠模型。结论成功建立了可用于评价NS3/4A蛋白酶的可视化小鼠模型。     

A novel screen-printed microfluidic paper-based electrochemical device for detection of glucose and uric acid in urine

A novel screen-printed microfluidic paper-based analytical device with all-carbon electrode-enabled electrochemical assay (SP-ACE-EC-μPAD) has been developed. The fabrication of these devices involved wax screen-printing, which was simple, low-cost and energy-efficient. The working, counter and reference electrodes were screen-printed using carbon ink on the patterned paper devices. Different wax screen-printing processes were examined and optimized, which led to an improved method with a shorter heating time (~5 s) and a lower heating temperature (75 °C). Different printing screens were examined, with a 300-mesh polyester screen yielding the highest quality wax screen-prints. The carbon electrodes were screen-printed on the μPADs and then examined using cyclic voltammetry. The analytical performance of the SP-ACE-EC-μPADs for the detection of glucose and uric acid in standard solutions was investigated. The results were reproducible, with a linear relationship [R²?=?0.9987 (glucose) or 0.9997 (uric acid)] within the concentration range of interest, and with detection limits as low as 0.35 mM (glucose) and 0.08 mM (uric acid). To determine the clinical utility of the μPADs, chronoamperometry was used to analyze glucose and uric acid in real urine samples using the standard addition method. Our devices were able to detect the analytes of interest in complex real-world biological samples, and have the potential for use in a wide variety of applications.     

一种离体皮肤经皮电阻测量装置的研制

目的 研制一种能固定离体皮肤和稳定测量经皮电阻值的离体皮肤经皮电阻测量装置.方法 离体皮肤经皮电阻测量装置由离体皮肤固定装置和经皮电阻测量仪组成,皮肤固定装置固定离体皮肤,固定装置装有电解质溶液,皮肤与内外之间的电解质溶液形成电导池,电阻测量仪通过皮肤固定装置中的内外电极,测量离体皮肤的经皮电阻值.取同一只Wistar大鼠背侧部的8块正常离体皮肤固定于离体皮肤固定装置中,测量并记录18个时间点的经皮电阻值,以上实验重复3次.结果 检测结果在正常值范围内,3次重复实验的经皮电阻值变化曲线基本一致,说明该装置能维持离体皮肤在24 h内屏障功能的稳定.结论 离体皮肤经皮电阻测量装置能满足皮肤刺激性和（或）腐蚀性试验离体皮肤替代方法的实验要求,还能用于其他有关药物的释放和透皮吸收试验的体外方法,以及测量经皮电阻值的相关方法.     

Hydrophilic electrospun polyurethane nanofiber matrices for hMSC culture in a microfluidic cell chip

Mimicking cellular microenvironments by MEMS technology is one of the emerging research areas. Integrated biomimetic systems with nanofiber polymer networks and microfluidic chips were fabricated and cellular behaviors were observed by changing surface characteristics of nanofibers and flow rates of microchannels. Modification of polyurethane nanofiber surfaces were achieved by grafting acrylic acid with plasma treatment and these nanofiber matrices were employed in a poly(dimethylsiloxane) based microfluidic chip. The surface characteristics of both electrospun nanofiber matrices was evaluated by measuring contact angle, porosity, and chemical structure using attenuated total reflection-Fourier transform infrared spectrometry. After modification, a terminal carboxyl group formed on the nanofiber surface and the wettability increased significantly. Human MSCs were seeded on the nanofiber matrices and a morphological investigation with actin filament staining and scanning electron microscopy was performed. A proliferation test by WST-1 and Live/Dead assay were performed to investigate the cell culture environment. It was observed that the cells on the AA-grafted nanofibers spread and proliferate compared to untreated nanofibers. It has also shown that flow rates in the microchannels played an important role for cell proliferation (Sim et al., Lab Chip 2007;7:1775-1782). Integration of nanofiber matrices into the microchannels provides the useful tools for mimicking cellular microenvironments and elucidating basic questions of cell and ECM assembly and interactions.     

Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices

Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5–8.5) and pre-incubation at low temperature (～4 °C) before polymerization at 37 °C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening.