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The response of two well-characterized human breast cancer cell lines (MCF-7 and MDA-MB-231) to a series of nutrient deficiencies is investigated with a label-free cell assay platform. The motivation of the research is to analyze adaptive responses of tumor cell metabolism and to find limiting conditions for cell survival. The platform measures extracellular values of pH and dissolved oxygen saturation to provide data of extracellular acidification rates and oxygen uptake rates. Additional electric cell substrate impedance sensing and bright-field cell imaging supports the data interpretation by providing information about cell morphological parameters. A sequential administration of nutrient depletions does not cause metabolic reprogramming, since the ratios of oxygen uptake to acidification return to their basal values. While the extracellular acidification drops sharply upon reduction of glucose and glutamine, the oxygen uptake is not affected. In contrast to other published data, cell death is not observed when both glucose and glutamine are depleted and cell proliferation is not inhibited, at least in MCF-7 cultures. It is assumed that residual concentrations of nutrients from the serum component are able to maintain cell viability when delivered regularly by active flow like in the cell assay platform, and, in a similar way, under physiological conditions.

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Flow-induced shear stress on adherent cells leads to biochemical signaling and mechanical responses of the cells. To determine the flow-induced shear stress on adherent cells cultured in a micro-scaled reaction chamber, we developed a suitable finite element method model. The influence of the most important parameters-cell shape, cell density, shear modulus and fluid velocity-was investigated. Notably, the cell shape strongly influences the resulting shear stress. Long and smooth cells undergo lower shear stress than more rounded cells. Changes in the curvature of the cells lead to stress peaks and single cells experience higher shear stress values than cells of a confluent monolayer. The computational results of the fluid flow simulation were validated experimentally. We also analyzed the influence of flow-induced shear stress on the metabolic activity and shape of L929, a mouse fibroblast cell line, experimentally. The results indicate that threshold stress values for continuous flow conditions cannot be transferred to quasi static flow conditions interrupted by short fluid exchange events.

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The capability of continuously monitoring cells and tissues in real-time for hours or days supports the value of a sensor-based microphysiometric approach. While there are widespread applications now for in-vitro settings, the use for smart, implanted devices is just beginning. The spectrum of analysed functional parameters comprises cellular morphological dynamics, metabolic activity and patterns of electric activity. An outline of a study on human tumor tissue samples gives an example of the potential benefits and challenges of in-vitro microphysiometry.

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The Intelligent Mobile Lab (IMOLA) delivers metabolic and morphological parameters of living cells in a label-free and real time way. It represents a key technology for the development of new cell-based assays. Electrochemical microsensors are used to measure the extracellular acidification (pH), cellular respiration (pO2), changes in cell number and morphology (electric impedance) in a controlled environment. These parameters are closely linked to the intracellular signaling network of the living cells. They are thus likely to respond sensitively to changes in cellular vitality. A wide spectrum of cell types can be tested with the system, including adherent and suspended cells, continuous cell lines, primary cells or tissue samples. The platform is described in detail and applications in the field's oncology, toxicology and environmental monitoring are shown.

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Cellular assays have become a fundamental technique in scientific research, pharmaceutical drug screening or toxicity testing. Therefore, the requirements of technical developments for automated assays raised in the same rate. A novel measuring platform was developed, which combines automated assay processing with label-free high-content measuring and real-time monitoring of multiple metabolic and morphologic parameters of living cells or tissues. Core of the system is a test plate with 24 cell culture wells, each equipped with opto-chemical sensor-spots for the determination of cellular oxygen consumption and extracellular acidification, next to electrode-structures for electrical impedance sensing. An automated microscope provides the optical sensor read-out and allows continuous cell imaging. Media and drugs are supplied by a pipetting robot system. Therefore, assay can run over several days without personnel interaction. To demonstrate the performance of the platform in physiologic assays, we continuously recorded the kinetics of metabolic and morphologic parameters of MCF-7 breast cancer cells under the influence of the cytotoxin chloroacetaldehyde. The data point out the time resolved effect kinetics over the complete treatment period. Thereby, the measuring platform overcomes problems of endpoint tests, which cannot monitor the kinetics of different parameters of the same cell population over longer time periods.

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Living cultured cells react to external influences, such as pharmaceutical agents, in an intricate manner due to their complex internal signal processing. Impedance sensing of cells on microelectrodes is a favored label-free technology to indicate cellular events, usually ascribed to morphologic alteration or changes in cellular adhesion, which is usually found in stand-alone systems that do not incorporate life support or additional sensor systems. However, only in symbiosis with metabolic activity sensing and picture documentation may a complete insight into cellular vitality be provided. This complement was created within the framework of an automated high-content screening system previously developed by our group, monitoring 24 cell culture chambers in parallel. The objective of this paper is the development of miniaturized electronics for impedance measurements and its system integration as a modular unit. In addition, it is shown how sensor electrodes were optimized by impedance matching such that spectroscopy and raw data analysis become feasible for every culture well. Undesired mechanical stress on cultured cells may arise from the medium and agent support system of the autonomous screening apparatus. This paper demonstrates how this hazard is treated with the simulation of microfluidics and impedance measurements. Physiological data are subsequently derived from the exemplary tumor cell line MCF-7 both during treatment with the agent doxorubicin and through the impact of natural killer cells. This correlates the information content of complex impedance spectra with cellular respiration as well as data from microscopy.

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In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.

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PURPOSE: To study the interplay of drugs and energy metabolism of tumor cells, metabolic changes induced by chloroacetaldehyde and cytochalasin B were analyzed in colon carcinoma cells LS174T. METHODS: O(2)-consumption and extracellular acidification were recorded using a bioelectronic sensor-chip system, which monitors these parameters in a culture continuously for at least 24 h. In parallel cultures cell number, cellular ATP-content, mitochondrial transmembrane potential, and the content of reactive oxygen species (ROS) were determined. RESULTS: When cell death was induced by chloroacetaldehyde (50 muM), the rate of acidification declined gradually for the next 15 h, while O(2)-consumption decreased rapidly within 30 min. This correlated with a loss in mitochondrial potential. However, cellular ATP-level showed a transient increase at 2 h; also ROS levels increased up to 6 h. In cells treated with cytochalasin B (2 muM), which inhibits glucose uptake, the rate of O(2)-consumption increased and the acidification activity dropped, even upon glutamine depletion. Mitochondrial membrane potential transiently increased after 1 h, while ATP-content decreased; there was no change in the level of ROS. CONCLUSION: The pattern of changes in basic energy metabolism differs with the type of cell death and growth inhibition involved in the cytotoxic action of two different drugs.

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Multiparametric silicon sensor chips mounted into biocompatible cell culture units have been used for investigations on cellular microphysiological patterns. Potentiometric, amperometric and impedimetric microsensors are combined on a common cell culture surface on the chip with an area of approximately 29 mm2. Extracellular acidification rates (with pH-sensitive field effect transistors, ISFETs), cellular oxygen consumption rates (with amperometric electrode structures) and cell morphological alterations (with impedimetric electrode structures, IDES) are monitored on single chips simultaneously for up to several days. The corresponding test device accommodates six of such sensor chips in parallel, provides electronic circuitry and maintains the required cell culture conditions (temperature, fluid perfusion system). Sensor data are transformed into quantitative information about microphysiologic conditions. The outcome of this transformation as well as reliability and sensitivity in detection of drug effects is discussed. This is the first report on multiparametric cell based assays with data obtained solely with integrated sensors on silicon chips. Those assays are required in different fields of application such as pharmaceutical drug screening, tumor chemosensitivity tests and environmental monitoring.

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In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.

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Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinician's decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

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The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.

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The pH in the cellular microenvironment (pH(M)) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pH(M) close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pH(M) of few (five to ten) adherent tumor cells in a distance of 10-100 nm from the cell plasma membrane. Experiments showed a pH(M)-value of 6.68 +/- 0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

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Critical parameters to be assessed in cell culture are the number of viable cells and cell viability. Growth, product formation, toxicity effects and the overall success of cell culture can depend largely on these. With interdigitated electrode structures (IDES) adherent cells are cultured directly on a pair of interdigitated electrodes, and the impedance of the system gives insight into the adhesive behaviour of the cells. The signal is influenced by the changes in number, growth and morphological behaviour of adherently growing cells, mainly owing to the insulating effects of the cell membranes. Five different cell lines are used, and their divergent behaviour is monitored over a period of four days, from inoculation of the cells to killing of the cells at the end of the experiments. Even when the cells from close monolayers, great fluctuations in the impedance signal can be observed. Nevertheless, for a more complete description of cellular systems, other parameters, such as acidification and respiration, have to be included in the measuring system.

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Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

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Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. The outstanding benefit lies in the passive behaviour of continuously working transducers, which in turn allows the dynamic recording of function-specific cellular processes. The microsensor system presented in this paper is a modular arrangement of various planar and non-planar sensor elements surrounding small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it proved to be an effective device both for toxicological and environmental research.

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A new method for on-line and real-time monitoring of concentration, growth and physiological state of cells in culture is described. This biosensor is based on impedance measurements of adherently growing cells on interdigitated electrode structures (IDES). The measurements can be performed for several days as there is no detectable electrical influence on the cells. The versatility of this new sensor is shown with some exemplary experiments. Cell density, growth and long-term behaviour of cells on the electrodes clearly change the impedance of the IDES. Both, the global influence of serum components (deprivation of foetal bovine serum) and the toxic effects of heavy metal ions (cadmium) result in changes of the sensor signal and can be visualized.

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The development of an integrated sensor system, called the physiocontrol microsystem, is presented. It is suited for microscopy, and works with both adherent cell types and cultures growing in suspension, as well as with tissue biopsies. The central part, a miniaturized culture chamber equipped with differently constructed microsensors, allows continuous observation of important physiological parameters even in the course of long-lasting experiments. Besides a description of the physical components, the study provides a summary of selected applications of the physiocontrol microsystem in basic cellular research and biomedical diagnostics.

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