An automatic and quantitative on-chip cell migration assay using self-assembled monolayers combined with real-time cellular impedance sensing

Cell migration is crucial in many physiological and pathological processes including embryonic development, immune response and cancer metastasis. Traditional methods for cell migration detection such as wound healing assay usually involve physical scraping of a cell monolayer followed by an optical observation of cell movement. However, these methods require hand-operation with low repeatability. Moreover, it's a qualitative observation not a quantitative measurement, which is hard to scale up to a high-throughput manner. In this article, a novel and reliable on-chip cell migration detection method integrating surface chemical modification of gold electrodes using self-assembled monolayers (SAMs) and real-time cellular impedance sensing is presented. The SAMs are used to inhibit cell adherence forming an area devoid of cells, which could effectively mimic wounds in a cell monolayer. After a DC electrical signal was applied, the SAMs were desorbed from the electrodes and cells started to migrate. The process of cell migration was monitored by real-time impedance sensing. This demonstrates the first occurrence of integrating cellular impedance sensing and wound-forming with SAMs, which makes cell migration assay being real-time, quantitative and fully automatic. We believe this method could be used for high-throughput anti-migratory drug screening and drug discovery.     

Real-time characterization of cytotoxicity using single-cell impedance monitoring

Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.     

二水草酸钙的形成及其对尿路结石的防治

二水草酸钙(COD)是泌尿系结石的主要成分之一,其成核、生长和聚集过程与尿石的形成密切相关。本文结合我们近年来的工作,综述了生物膜及其模拟膜对COD的调控作用,尿大分子、焦磷酸盐、多磷酸盐、柠檬酸、酒石酸及表面活性剂等尿小分子的诱导作用,过饱和度、化学计量条件、pH值、离子强度、温度等体系参数对COD形成的影响;讨论了COD的形貌、晶面电荷及其与细胞膜粘附的研究进展。从临床上预防和治疗草酸钙结石的角度,综述了有利于COD形成的因素。     

肾上皮细胞损伤使草酸钙晶体黏附增强的分子机制

研究了非洲绿猴肾上皮细胞(Vero)在损伤前后与一水合草酸钙(COM)和二水合草酸钙(COD)晶体的黏附作用及其引起的细胞反应,探讨了肾结石形成机理.COM和COD晶体与损伤细胞的黏附加重了细胞的过氧化损伤程度,导致损伤细胞的活力进一步降低,乳酸脱氢酶(LDH)释放量和活性氧(ROS)进一步增加,坏死细胞数量进一步增多,细胞体积缩小,并出现凋亡小体.COM晶体对细胞的损伤能力显著大于COD晶体.扫描电子显微镜(SEM)观测结果表明,损伤组Vero与COM微晶的黏附作用显著强于对照组,且能促进COM微晶的聚集.共聚焦显微镜观测结果表明,Vero损伤后,其表面表达的晶体黏附分子透明质酸(HA)显著增加,HA分子是促进微晶黏附的重要原因.细胞表面草酸钙的黏附量和晶体聚集程度与细胞的损伤程度成正相关.本文结果从分子和细胞水平上提示,细胞损伤是导致草酸钙肾结石形成的重要因素.     

AFM study of hippocampal cells cultured on silicon wafers with nano-scale surface topograph

The rat hippocampal cells were selected as model to study the interaction between the neural cells and silicon substrates using atomic force microscopy (AFM). The hippocampal cells show tight adherence on silicon wafers with nano-scale surface topograph. The lateral friction force investigated by AFM shows significant increase on the boundary around the cellular body. It is considered to relate to the cytoskeleton and cellular secretions. After ultrasonic wash in ethanol and acetone step by step, the surface of silicon wafers was observed by AFM sequentially. We have found that the culture leftovers form tight porous networks and a monolayer on the silicon wafers. It is concluded that the leftovers overspreading on the silicon substrates are the base of cell adherence on such smooth inert surfaces.     

Newly developed chemical probes and nano-devices for cellular analysis.

A cellular analyzing system including a "real-time cellular imaging system" and a "comprehensive analyzing system for cellular responses" was developed. A "real-time cellular imaging system" is a system used to measure real-time imaging of multiple phenomena of a single cell with high special and temporal resolutions for the purpose to understand the pathology and physiology in a single cell and realize to single cell level diagnosis. A "real-time cellular imaging system" includes multi-probe imaging with AFM (atomic force microscopy), optical and SECM (scanning electrochemical microscopy) modes, which provides us with topological information and biochemical reactions at the local area of the interior and exterior of a cell. Scanning electrochemical/optical microscopy was applied to image PC12 cells. On the other hand, cells respond to their specific substances via their ligands. Therefore, the comprehensive analysis of protein-protein interaction is the important issue to determine the functions of cells. For this purpose, a "comprehensive analysis system for cellular responses" was developed. This system is based on SPR (surface plasmon resonance) and MS (mass spectrometry) using a nano-fabricated substrate. The interaction between IL-1 beta and anti-IL-1 beta antibodies was detected.     

Promotion on Nucleation and Aggregation of Calcium Oxalate Crystals by Injured African Green Monkey Renal Epithelial Cells

The purpose of this work was to detect the properties of African green monkey renal epithelial cells (Vero) after oxidative injury and to study the mediation of the injured Vero on aggregation and formation of calcium oxalate crystals. This injury model was induced by 0.15 mmol/L H₂O₂ according to the pretest evaluation. The results suggested that H₂O₂ could injure Vero significantly and decrease cell viability in a time‐dependent manner for exposure time of 0.5–2 h. After cell injury, the indexes connected with oxidative injury changed. The malondialdehyde (MDA) content and osteopontin (OPN) expression increased, while superoxide dismutase (SOD) level decreased. It resulted in the increase of both the amount of CaOxa crystals and the degree of crystal aggregation on the injured cells. This work indicated that injured cells promoted the formation of calcium oxalate monohydrate (COM) crystals, thus increased the risk of formation of urinary stone.     

Recognition of chemical compounds in contaminated water using time-dependent multiple dose cellular responses

An early determination of toxicant compounds of water contaminations can gain critical time to protect citizens’ health and save substantial amounts of medical costs. To determine toxins in real time, a multi-dose classification algorithm using cellular state variable identification (CSVID) is developed in this paper. First, the dynamic cytotoxicity response profiles of living cells are measured using a real-time cell electronic sensing (RT-CES) system. Changes in cell number expressed as cell index (CI) are recorded on-line as time series. Then CSVID, which reflects the cell killing, cell lysis and certain cellular pathological changes, is extracted from those dynamic cellular responses. Finally, a support vector machine (SVM) algorithm based on CSVID is employed to classify chemical compounds and determine their analogous cellular response pathway. In order to increase the classification accuracy, a majority vote of the class labels is also proposed. Several validation studies demonstrate that CSVID-based classification algorithm has great potential in distinguishing the cytotoxicity response of the cells in the presence of toxins.     

High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells

The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell’s kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p?>?0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

Submicroparticles composed of amphiphilic chitosan derivative for oral insulin and curcumin release applications

Amphiphilic polymers for dual drug delivery have been a focus of research in recent years. We have previously developed and characterized Lauroyl sulphated chitosan (LSCS). Here biological characterizations like mucoadhesion, cytotoxicity, calcium binding, tight junction opening and enzymatic degradation studies were performed to understand its applicability. In vitro drug release properties of both hydrophilic insulin and hydrophobic curcumin were carried out. The biological activity and stability of released insulin were also studied. The stability studies of encapsulated curcumin and uptake studies have also been carried out. LSCS showed strong mucoadhesion and 100% of non-toxicity. LSCS could transiently open tight junctions between Caco-2 cells and thus increase the paracellular permeability. LSCS enhanced calcium binding properties and decreased enzymatic degradation rate retaining insulin activity. LSCS could protect curcumin from photodegradation and could also enter into the cells. From release studies, LSCS was found to be a suitable candidate for both drugs.     

植物药抑制泌尿系结石形成的化学基础

植物药治疗泌尿系结石具有独特的疗效.本文综述了国内外植物药(泽泻,Phyllanthus niruri,Zeamays,Agropyron repens和Herniaria hirsute等)在体外模拟实验和动物实验中对泌尿系结石形成的影响,植物药抑制泌尿系结石形成的机制和化学基础是:植物药与钙离子发生配位,降低尿石盐的过饱和度;抑制一水草酸钙生长,诱导二水草酸钙形成;抑制晶体生长和聚集;可以保护尿路粘膜,防止晶体在肾上皮细胞上发生粘附;改变尿石形成促进剂和抑制剂的排泄量.     

Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor

This paper describes a microquantitative method for glucose determination in situ of living cells in real-time. In this novel technique adherent cells are cultured onto microcarrier beads and packed into a renewable microcolumn within a microsequential injection lab-on-valve system (microSI-LOV). Glucose sensing is performed through the use of a two-step, NAD-linked enzymatic process. The course of the assay is monitored in real-time, by absorbance of NADH at 340 nm. The microsequential assay based on plug/nozzle design has a linear dynamic range for glucose of 0.1 to 5.6 mM. The design of the (microSI-LOV) system allows the assay to be carried out using only 40 microL of the enzyme reagent and 3 microL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopore beads. Rapid cellular glucose consumption, in this technique, is facilitated by a high cell density, which allows a large number of cells (10(4)-10(5)) to be retained in a very small volume (3 microL). In turn, this cell density results in the rapid depletion of glucose from the cell medium over short time periods (< 2 min). In conjunction with the assay development, the plug/nozzle design and its ramifications on mixing in general are presented and discussed.     

Ultrasmall Nanopipette: Toward Continuous Monitoring of Redox Metabolism at Subcellular Level

Ionic current rectification (ICR) based nanopipettes allow accurate monitoring of cellular behavior in single living cells. Herein, we proposed a 30 nm nanopipette functionalized with G‐quadruplex DNAzyme as an efficient biomimetic recognizer for ROS generation at subcellular level via the changes of current–voltage relationship. Taking advantages of the ultra‐small tip, the nanopipette could penetrate into a single living cell repeatedly or keep measuring for a long time without compromising the cellular functions. Coupled with precision nanopositioning system, generation of ROS in mitochondria in response to cell inflammation was determined with high spatial resolution. Meanwhile, the changes of aerobic metabolism in different cell lines under drug‐induced oxidative stress were monitored continuously. We believe that the ICR‐nanopipette could be developed as a powerful approach for the study of cellular activities via electrochemical imaging in living cells.     

High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 μm diameter, 8 μm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 μm diameter and 8 μm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 μM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.     

微流控芯片药物诱导细胞凋亡

发展了一种以微流控芯片为平台的药物诱导细胞凋亡的新方法.选择HeLa细胞为对象,通过浓度梯度芯片形成稳定的药物浓度梯度,诱导HeLa细胞凋亡,利用荧光能量共振转移（fluorescence resonance energy transfer,FRET）成像系统进行实时监测,分析细胞对不同浓度化合物的毒性反应.结果表明,细胞在顺铂诱导下发生明显的起泡和皱缩,FRET比率值逐渐降低,在药物浓度梯度作用下,芯片每个通道内细胞呈现不同程度的凋亡.该方法实现了药物浓度梯度诱导细胞凋亡的实时监测和定量分析,为抗肿瘤药物评价和高通量药物筛选提供了新的手段.     

玉米须提取液对尿液中草酸钙晶体形成的影响

本文采用X射线衍射(XRD)、红外光谱(FTIR)、扫描电子显微镜(SEM)等方法分析了玉米须提取液对正常人尿液中草酸钙晶体形成的影响,通过电导率法研究了草酸钙晶体生长的动力学过程,以及从生物矿化的角度对玉米须提取液影响尿液中草酸钙晶体的可能机理进行了探讨。由于玉米须提取液中有机酸或多糖的羟基、羰基等通过配位作用与Ca²⁺结合形成可溶性配位化合物,减少了Ca²⁺与Oxa^2-的结合能力,从而抑制了CaOxa的成核和生长。同时,可能由于玉米须提取液中有效成分与二水草酸钙(COD)的吸附点键合,增强了COD晶体在溶液中的热力学稳定性,进而抑制了COD晶体向热力学更稳定态的一水草酸钙(COM)晶体转变。结果显示,这种抑制作用随玉米须浓度增大而增大,且COD晶体尺寸随着玉米须浓度的增大而减小。玉米须抑制COD晶体向COM晶体转变的作用为开发预防和治疗尿结石的药物提供了启示。     

Tight junction development between cultured hepatoma cells: possible stages in assembly and enhancement with dexamethasone.

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: 1) "formation zones" (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; 2) linear strands and aggregates of 9--11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; 3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specializations may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 x 10(-6)M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

聚合物与草酸钙相互作用的研究进展及其对预防尿路结石的意义

聚合物对泌尿系结石的形成具有抑制和促进双重作用。本文综述了聚合物对尿石矿物草酸钙成核、生长、聚集及其与尿路细胞膜黏附影响的研究进展,讨论了聚合物影响草酸钙晶面和形貌的机理,指出了该领域所面临的问题和将来的发展方向。     

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel

Deciphering the signaling pathways that govern stimulation of na?ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na?ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.     

Deoxycholic Acid Modulates Cell-Junction Gene Expression and Increases Intestinal Barrier Dysfunction

Diet-related obesity is associated with increased intestinal hyperpermeability. High dietary fat intake causes an increase in colonic bile acids (BAs), particularly deoxycholic acid (DCA). We hypothesize that DCA modulates the gene expression of multiple cell junction pathways and increases intestinal permeability. With a human Caco-2 cell intestinal model, we used cell proliferation, PCR array, biochemical, and immunofluorescent assays to examine the impact of DCA on the integrity of the intestinal barrier and gene expression. The Caco-2 cells were grown in monolayers and challenged with DCA at physiological, sub-mM, concentrations. DCA increased transcellular and paracellular permeability (>20%). Similarly, DCA increased intracellular reactive oxidative species production (>100%) and accompanied a decrease (>40%) in extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. Moreover, the mRNA levels of 23 genes related to the epithelial barrier (tight junction, focal adhesion, gap junction, and adherens junction pathways) were decreased (>40%) in (0.25 mM) DCA-treated Caco-2 cells compared to untreated cells. Finally, we demonstrated that DCA decreased (>58%) the protein content of occludin present at the cellular tight junctions and the nucleus of epithelial cells. Collectively, DCA decreases the gene expression of multiple pathways related to cell junctions and increases permeability in a human intestinal barrier model.