Multi-sensor arrays for online monitoring of cell dynamics in in vitro studies with choroid plexus epithelial cells.

Sensors and multi-sensor arrays are the basis of new technologies for the non-label monitoring of cell activity. In this paper we show that choroid plexus cells can be cultured on silicon chips and that sensors register in real time changes in their activity, constituting an interesting experimental paradigm for cell biology and medical research. To validate the signals recorded (metabolism = peri-cellular acidification, oxygen consumption = respiration; impedance = adhesion, cell shape and motility) we performed experiments with compounds that act in a well-known way on cells, influencing these parameters. Our in vitro model demonstrates the advantages of multi-sensor arrays in assessment and experimental characterization of dynamic cellular events--in this case in choroid plexus functions, however with applicability to other cell types as well.

The surface topography of the choroid plexus. Environmental, low and high vacuum scanning electron microscopy.

Environmental scanning electron microscopy (ESEM) allows the examination of hydrated and dried specimens without a conductive metal coating which could be advantageous in the imaging of biological and medical objects. The aim of this study was to assess the performance and benefits of wet-mode and low vacuum ESEM in comparison to high vacuum scanning electron microscopy (SEM) using the choroid plexus of chicken embryos as a model, an organ of the brain involved in the formation of cerebrospinal fluid in vertebrates. Specimens were fixed with or without heavy metals and examined directly or after critical point drying with or without metal coating. For wet mode ESEM freshly excised specimens without any pre-treatment were also examined. Conventional high vacuum SEM revealed the characteristic morphology of the choroid plexus cells at a high resolution and served as reference. With low vacuum ESEM of dried but uncoated samples the structure appeared well preserved but charging was a problem. It could be reduced by a short beam dwell time and averaging of images or by using the backscattered electron detector instead of the gaseous secondary electron detector. However, resolution was lower than with conventional SEM. Wet mode imaging was only possible with tissue that had been stabilized by fixation. Not all surface details (e.g. microvilli) could be visualized and other structures, like the cilia, were deformed. In summary, ESEM is an additional option for the imaging of bio-medical samples but it is problematic with regard to resolution and sample stability during imaging.

The Bionas technology for anticancer drug screening.

BACKGROUND: The Bionas 2500(®) analyzing system is an advanced label-free technology using a cell-based multi-sensor array, which is commercially available. Data on metabolism, respiration, adhesion, cell proliferation and cell death rates, as well as ligand-receptor interactions (multi-parametric) can be acquired and statistically evaluated. Noteworthy is the possibility of analyzing later after effects and/or recovery after drug treatment. In addition, Bionas supports all conceivable drug application modes (one, two or more drugs). Specimens for drug screening with Bionas consist of human permanent cell lines, primary cell cultures as well as 'tumor slices' obtained from biopsies and surgery material. OBJECTIVE/METHODS: Examples of measurements are presented and discussed. CONCLUSION: Although drug throughput is modest, the high quality of the information allows in-depth evaluation.

A new method to assess drug sensitivity on breast tumor acute slices preparation.

A method for assessing tumor drug sensitivity is described that is based on preparation of tissue slices and use of silicon chips equipped with electrochemical sensors (multisensor array). The tumor slices (200-300 microM thick) are prepared after surgery and incubated in a medium for recovery after slicing. The advantage, compared to other preparations, is that the original three-dimensional structure is retained. Multisensor arrays measure: (a) pericellular acidification (anaerobic metabolism) and (b) oxygen consumption (respiration). The innovative aspect is that such measurements can be made online, as opposed to using a large battery of endpoint tests on cell vitality and proliferation. Electron microscopy of slices serves to determine cell density and structure and induction of apoptosis/necrosis. Slices of more than 200 breast tumors were used. Metabolic activity was inhibited by sodium fluoride, which reduces glycolysis, and potassium cyanide, which inhibits respiration. These changes are thus reflected in the curves of acidification and oxygen consumption. In other experiments the cytostatic Taxol, an anticytoskeletal agent, was used showing dose and time-dependent effects on acidification and oxygen consumption. In conclusion, the method presented here, is able to provide information on drug sensitivity of a tumor, which aids in designing individualized therapy and is used for drug screening.

Application of silicon sensor technologies to tumor tissue in vitro: detection of metabolic correlates of chemosensitivity.

Silicon sensor technologies, developed during the 1990s, allow measurement of extracellular chemical changes related to cell metabolism. Exposition of tumor cells in vitro to anticancer drugs modifies cell metabolism, making it possible to detect on-line with sensor chips patterns of metabolic activity, which depend on drug sensitivity, or drug resistance of the cells. Sensor devices are composed of an incubation chamber with a sensor chip and a fluidic system for medium supply. Basically, two sensor types are available: (1) monosensor systems to detect extracellular acidification; and (2) multisensor arrays for many parameters such as pH, oxygen consumption, and impedance. Two companies have developed such systems: Molecular Devices (USA) and Bionas (Germany). In this chapter, in addition to operation of the sensor devices, we describe techniques for tissue (tumor and non-tumor) preparation. Basically, three procedures are described: (1) tissue dissociation and further cultivation on the sensor chip or on Transwell inserts; (2) preparation of tissue slices (300 microm thick) and attachment to the sensor chip or to inserts, and (3) cultivation of cells in dialysis tubes, a procedure necessary for nonadherent cells and cell suspensions to avoid their washing away. Evaluation of results and selection of controls are also discussed.