共查询到20条相似文献，搜索用时 31 毫秒
1.
A method for monitoring the biological exocytotic phenomena on a microfluidic system was proposed. A microfluidic device coupled with functionalities of fluorescence imaging and amperometric detection has been developed to enable the real-time monitoring of the exocytotic events. Exocytotic release of single SH-SY5Y neuroblastoma cells was studied. By staining the cells located on integrated microelectrodes with naphthalene-2,3-dicarboxaldehyde, punctuate fluorescence consistent with localization of neurotransmitters stored in vesicles was obtained. The stimulated exocytotic release was successfully observed at the surface of SH-SY5Y cells without refitting the commercial inverted fluorescence microscope. Spatially and temporally resolved exocytotic events from single cells on a microfluidic device were visualized in real time using fluorescence microscopy and were amperometrically recorded by the electrochemical system simultaneously. This coupled technique is simple and is hoped to provide new insights into the mechanisms responsible for the kinetics of exocytosis. 相似文献
2.
Na Xu Zhen-Feng Zhang Li Wang Bo Gao Dai-Wen Pang Han-Zhong Wang Zhi-Ling Zhang 《Biomicrofluidics》2012,6(3)
Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection. 相似文献
3.
High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment 总被引：1，自引：0，他引：1
Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis. 相似文献
4.
Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments 总被引：1，自引：0，他引：1
Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory. 相似文献
5.
A combination of a microfluidic device with a light modulation system was developed to detect the oxygen consumption rate (OCR) of a single developing zebrafish embryo via phase-based phosphorescence lifetime detection. The microfluidic device combines two components: an array of glass microwells containing Pt(II) octaethylporphyrin as an oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids above the microwells to controllably seal the microwells of interest. The total basal respiration (OCR, in pmol O2/min/embryo) of a single developing zebrafish embryo inside a sealed microwell has been successfully measured from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage (48 hpf). The total basal respiration increased in a linear and reproducible fashion with embryonic age. Sequentially adding pharmacological inhibitors of bioenergetic pathways allows us to perform respiratory measurements of a single zebrafish embryo at key developmental stages and thus monitor changes in mitochondrial function in vivo that are coordinated with embryonic development. We have successfully measured the metabolic profiles of a single developing zebrafish embryo from 3 hpf to 48 hpf inside a microfluidic device. The total basal respiration is partitioned into the non-mitochondrial respiration, mitochondrial respiration, respiration due to adenosine triphosphate (ATP) turnover, and respiration due to proton leak. The changes in these respirations are correlated with zebrafish embryonic development stages. Our proposed platform provides the potential for studying bioenergetic metabolism in a developing organism and for a wide range of biomedical applications that relate mitochondrial physiology and disease. 相似文献
6.
Hui Huang Lili Jiang Shu Li Jun Deng Yan Li Jie Yao Biyuan Li Junsong Zheng 《Biomicrofluidics》2014,8(1)
Molecular gradients play a significant role in regulating biological and pathological processes. Although conventional gradient-generators have been used for studying chemotaxis and axon guidance, there are still many limitations, including the inability to maintain stable tempo-spatial gradients and the lack of the cell monitoring in a real-time manner. To overcome these shortcomings, microfluidic devices have been developed. In this study, we developed a microfluidic gradient device for regulating neuron axon guidance. A microfluidic device enables the generation of Brain-derived neurotrophic factor (BDNF) gradient profiles in a temporal and spatial manner. We test the effect of the gradient profiles on axon guidance, in the BDNF concentration gradient axon towards the high concentration gradient. This microfluidic gradient device could be used as a powerful tool for cell biology research. 相似文献
7.
Racetubes, a conventional system employing hollow glass tubes, are typically used for monitoring circadian rhythms from the model filamentous fungus, Neurospora crassa. However, a major technical limitation in using a conventional system is that racetubes are not amenable for real-time gas perturbations. In this work, we demonstrate a simple microfluidic device combined with real-time gas perturbations for monitoring circadian rhythms in Neurospora crassa using bioluminescence assays. The developed platform is a useful toolbox for investigating molecular responses under various gas conditions for Neurospora and can also be applied to other microorganisms. 相似文献
8.
The physiology of vascular endothelial cells is strongly affected by fluid shear stress on their surface. In this study, a microfluidic assay was employed to analyze the alignment of actin filaments in endothelial cells in response to shear stress. When cells were cultured in microfluidic channels and subjected to shear stress, the alignment of filaments in the channel direction was significantly higher than in static cultures. By adding inhibitory drugs, the roles of several signaling proteins in the process of alignment were determined. Thus, it is shown how microfluidic technology can be employed to provide a mechanistic insight into cell physiology. 相似文献
9.
Chao Wei Beiyuan Fan Deyong Chen Chao Liu Yuanchen Wei Bo Huo Lidan You Junbo Wang Jian Chen 《Biomicrofluidics》2015,9(1)
This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes. 相似文献
10.
Francesco Piraino ?eila Selimovi? Marco Adamo Alessandro Pero Sam Manoucheri Sang Bok Kim Danilo Demarchi Ali Khademhosseini 《Biomicrofluidics》2012,6(4)
The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10 × 10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to ∼1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies. 相似文献
11.
Danny Bavli Elishai Ezra Daniel Kitsberg Margarita Vosk-Artzi Shashi K. Murthy Yaakov Nahmias 《Biomicrofluidics》2016,10(2)
Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation. 相似文献
12.
Jiaqing Yu Ding Tao Ee Xing Ng Chester L. Drum Ai Qun Liu Chia-Hung Chen 《Biomicrofluidics》2014,8(5)
Thrombin, which has the leading role in the blood coagulation cascade, is an important biomarker in hemostasis and cardiovascular disease (CVD) development. In this study, a measurement system capable of continuously monitoring individual thrombin generation using droplet microfluidic technology is manipulated. The thrombin generation assay based on fluogenic substrate is performed within the droplets and the thrombin generation curve of plasma sample activated by tissue factor is measured in real-time to reflect the sample conditions dynamically. The injection of the inhibitor of thrombin generation is developed to assay the inhibited curve which relates to thrombin self-inhibition in biological systems. This microfluidic system is integrated with the microdialysis probe, which is useful to connect to the living animals for future in vivo real time thrombin measurements for rapid CVD diagnosis. 相似文献
13.
Malaria-infected red blood cells (iRBCs) become less deformable with the progression of infection and tend to occlude microcapillaries. This process has been investigated in vitro using microfluidic channels. The objective of this paper is to provide a quantitative basis for interpreting the experimental observations of iRBC occlusion of microfluidic channels. Using a particle-based model for the iRBC, we simulate the traverse of iRBCs through a converging microfluidic channel and explore the progressive loss of cell deformability due to three factors: the stiffening of the membrane, the reduction of the cell''s surface-volume ratio, and the growing solid parasites inside the cell. When examined individually, each factor tends to hinder the passage of the iRBC and lengthen the transit time. Moreover, at sufficient magnitude, each may lead to obstruction of narrow microfluidic channels. We then integrate the three factors into a series of simulations that mimic the development of malaria infection through the ring, trophozoite, and schizont stages. These simulations successfully reproduce the experimental observation that with progression of infection, the iRBC transitions from passage to blockage in larger and larger channels. The numerical results suggest a scheme for quantifying iRBC rigidification through microfluidic measurements of the critical pressure required for passage. 相似文献
14.
15.
We discuss the ability to perform fluorescent immunocytochemistry, following cell fixation, using a microfluidic array of primary, nonadherent, single CD34+ stem cells. The technique requires small cell samples and proceeds with no cell loss, making it well-suited to monitoring these rare patient-derived cells. The chip allows us to correlate live cell dynamics across arrays of individual cells with post-translational modifications of intracellular proteins, following their exposure to drug treatments. Results also show that due to the microfluidic environment, the time scale of cell fixation was significantly reduced compared to conventional methods, leading to greater confidence in the status of the protein modifications studied. 相似文献
16.
The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz–1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m2 (n = 23); NB4: 22.5 ± 4.7 mF/m2 (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach. 相似文献
17.
Elisa Cimetta Mauro Franzoso Marta Trevisan Elena Serena Alessandro Zambon Stefano Giulitti Luisa Barzon Nicola Elvassore 《Biomicrofluidics》2012,6(2):024127-12
Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time. 相似文献
18.
Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro. 相似文献
19.
Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development. 相似文献
20.
Viral infections remain a major threat to public health. The speed with which viruses are evolving drug-resistant mutations necessitates the further development of antiviral therapies with a large emphasis on drug discovery. To facilitate these efforts, there is a need for robust, high-throughput assays that allow the screening of large libraries of compounds, while enabling access to detailed kinetic data on their antiviral activity. We report here the development of a droplet-based microfluidic platform to probe viral fusion, an early critical step in infection by membrane-enveloped viruses such as HIV, Hepatitis C, and influenza. Using influenza A, we demonstrate the measurement of the kinetics of fusion of virions with target liposomes with sub-second temporal resolution. In analogy with acidification of the endosome that triggers fusion in a cellular context, we acidify the content of aqueous droplets containing virions and liposomes in situ by introducing acid from the dispersed phase and visualize the kinetics of fusion by using fluorescent probes. 相似文献