Cell-based biosensors and their application in biomedicine.

https://www.mdpi.com/2079-6374/2/2/127/pdf

Microfabricated electrochemical cell-based biosensors for analysis of living cells in vitro.

PurposeTo improve orthopedic implant ingrowth, knowledge of the effect of chemical surface modifications on vital cell function in vitro is of importance. Early in our investigations we recognized ...

Time-Dependent Metabolic Activity and Adhesion of Human Osteoblast-Like Cells on Sensor Chips with a Plasma Polymer Nanolayer:

Goal: This contribution points out the need for well-defined and documented data processing protocols in microphysiometry, an evolving field of label-free cell assays. The sensitivity of the obtained cell metabolic rates toward different routines of raw data processing is evaluated. Methods: A standard microphysiometric experiment structured in discrete measurement intervals was performed on a platform with a pH- and O 2-sensor readout. It is evaluated using three different data evaluation protocols, based on A) fast Fourier transformation of such dynamics, B) linear regression (LIN) of pH(t) and O2(t) dynamics, and C) numerical simulation (SIM) with a subsequent fitting of dynamics for parameter estimation. Results: We propose a sequence of well documented steps for an organized processing of raw sensor data. Figures of merit for the quality of raw data and the performance of data processing are provided. To estimate metabolic rates, a reaction-diffusion modeling approach is recommended if the necessary model input parameters such as the distribution of the active biomass, sensor response time, and material properties are available. Conclusion: The information about cellular metabolic activity contained by measured sensor data dynamics is superimposed by manifold sources of error. Careful consideration of data processing is necessary to eliminate these errors as much as possible and to avoid an incorrect interpretation of data.

Data Processing in Cellular Microphysiometry

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.

http://iopscience.iop.org/article/10.1088/0967-3334/36/7/1367/pdf

Nutrient depletion and metabolic profiles in breast carcinoma cell lines measured with a label-free platform.

In research, pharmacological drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells persists. As they are very sensitive, cells are used as signal transducers. For this purpose our research group has developed an automated high-content online measurement system, which is described in this paper. The system is comprised of a pipetting robot sensors for measurement of pH, pO2, electric signals and cell adhesion, a digital microscope and a climate chamber. The robot is used to supply the cells with nutrient solution or active agents that are to be tested, while the sensors monitor the acidification of the medium, the uptake of oxygen and the adhesion of the cells to the substrate. Reflected light as well as fluorescence imaging during the course of an experiment are possible due to the included microscope. Cells and sensors are arranged in a special 24-well micro plate that is placed at a fixed position within the system. Oxygen and pH values are usually measured every 5 to 15 seconds. The running software also allows planning of experiments over a long period of time (several days), which makes the system suitable for applications like pharmaceutical drug screening, in vitro chemo-sensitivity assays, toxicological studies and analytical histopathology.

Automated multi-parametric label free 24 channel real-time screening system

Using modeling and simulation, we quantify the influence of spatiotemporal dynamics on the accuracy of data obtained from sensors placed in microscaled reaction volumes. The model refers to cellular reaction (i.e. proton extrusion and oxygen consumption) in complex, buffering solutions. Whole cells or viable tissues cultured in such devices are monitored in real time with integrated sensors for pH and dissolved oxygen. A 3D finite element model of diffusion and metabolic reaction was set up. With respect to pH, the effect of buffering species on proton diffusion is analysed in detail. To account for the delayed time response of real sensors, the sensor impulse response time was implemented by linear convolution. A validation of the model has been achieved by an electrochemical approach. The model reveals significant deviations of measured pH and O2, and values of these parameters actually occurring at different sites of the cell culture volume. It is applicable to any setting of (bio-) sensors involving reaction and diffusion of dissolved gases and particularly H+ ions in buffered solutions.

Reaction–diffusion modelling for microphysiometry on cellular specimens

Zell-Chipsysteme - Mikrosensorarrays für die biologische Grundlagenforschung und Diagnostik

For monitoring of the metabolic and morphologic activity of living cells a six channel version of a cell based assay called intelligent mobile lab (IMOLA) was prepared (Wiest et al., 2006). IMOLA employs biochips which contain microsensors for measurement of cellular respiration, extracellular acidification and changes in morphology of the cells. These parameters are monitored in the microenvironment of living cells using sensors for dissolved oxygen, pH and impedance. Due to a sophisticated system set-up the cells can be monitored label-free, parallel, continuously and in real-time. The measurement is performed in a stopand go-mode. During the stop-mode (7 min) the microsensors detect the slope of oxygen consumption, acidification and morphological changes of the cells. Afterwards during a pump cycle (go-mode) cell culture media or a drug are transported toward the cells to supply them with fresh media and to recalibrate the microsensors. These cycles are repeated throughout the measurement which may last up to two weeks. In the presented experiment the toxicity of doxcorubicin toward MCF-7 cells compared to a control was investigated. The vitality of the cells is reduced for 33% compared to the control after 30 h of exposition. To ensure validity of the data the experiment was stopped by adding Triton X-100. Here the vitality signals of both groups declined immediately. With the presented IMOLA system a new tool for dynamic investigation of vitality and morphology of living cells and their interaction with drugs, toxins or metabolites is available. One advantage of the system is the label-free and long term investigation method. Due to these properties it is possible to monitor the kinetic of the interactions between cells and toxins. With this cell based assay, unique in-vitro toxicity data can be determined without the use of animal experiments.

Cell based assay for label-free, long-term investigation of living cells as alternative testing method for toxicity

There is an increasing demand for cellular screening systems. For this purpose our research group has developed an automated high-content online measurement system for living cells. The system is comprised of a pipetting robot, sensors for measurement of pH, pO 2 , electric signals and cell adhesion, a digital microscope and a climate chamber. The robot is used for media supply, while the sensors monitor the metabolic and morphological parameters of the cells. Reflected light and fluorescence imaging during the course of an experiment are possible due to the included microscope. Sensors are integrated in a special 24-well micro plate that is placed at a fixed position within the system. Oxygen and pH values are usually measured every 5 to 15 seconds. Experiments over a long period of time (several days) are possible, making the system suitable for applications like pharmaceutical drug screening, in vitro chemosensitivity assays, toxicological studies and analytical histopathology.

Real-time screening system using living cells for chemosensitivity testing

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