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Cell Profiling Assays
A new multiparametric, label-free and non-invasive solution
Overview
We present a new biosensor system that simultaneously measures three metabolically relevant parameters, such as oxygen consumption of test cells, the extra cellular acidification rate as well as cell adhesion, for cytological profiling of living cells.
The readout is continuous for up to several days. The technology, which can be used with many different cell types, including primary cell cultures, is easy to use and allows non-invasive and continuous observation of the effect of a test substance on the cells' metabolism. The sensor system is label-free, meaning cells are not affected by the actual measurements since no cell staining or labeling is involved. Concentration dependence or regeneration effects can also be investigated.
Cytological Profiling
The sensor system can detect a drugs' influence on cellular metabolism by measuring the acidification rate, the depletion of oxygen in the medium and cell adhesion. Activation of cellular metabolism is associated with increased glucose turnover and oxygen consumption, and with increased excretion of acidic breakdown products. Similarly, inhibition of metabolism is accompanied by a reduction in the excretion of acidic breakdown products and usually a reduction in oxygen consumption. In addition, inhibition with adhering cells is mostly associated with a change in attachment behavior or membrane stability.
Target Validation and Phenotypic Screening
The sensor system is useful for target validation studies and phenotypic screening of compounds in cells without knowing their target and assaying for varying effects such as proliferation, apoptosis and altered cellular metabolism.
The biosensor helps identify compounds that enhance or inhibit the abnormal glycolytic bias of tumor cells, or that modulate fatty acid oxidation (obesity) and glycolysis and respiration (diabetes). Finally, the sensor system can be applied to identify compounds against diseases that affect immune functions.
Prediction of Hepatoxicity Liabilities
Standardized HepG2 cells and non-immortalized primary human hepatocytes have been shown to be ideal biosensors for the analysis of lead compounds. The online and real-time observation of hepatoxicity liabilities with the Bionas® 2500 analyzing system has distinct advantages over current assays, which become apparent when comparing primary human hepatocytes and primary rat hepatocytes.
Rat and human test cell lines were cultivated in special medium without carbonate buffer on collagen coated sensor chips. Paracetamol (acetaminophen (AAP): IC50 19 mM), which is known for its liver toxicity, served as the test substance. Figure 1 first shows an initial run in medium without paracetamol (Running medium, RM). After about three hours (3 h) 1 mg/ml of paracetamol (compound) is added. Shown here are the acidification (red), respiratory rate (blue) and adhesion (green) of primary hepatocytes from rats and humans. Whereas only the respiratory activity is reduced in human hepatocytes, the rat hepatocytes clearly show stronger effects. Not only is the respiratory rate faster and more strongly reduced, but also the acidification rate is significantly inhibited even during the first 30 minutes of exposing rat hepatocytes to paracetamol. In addition, adhesion of the cells to the surface slowly but continuously declines.
Figure 1: Primary hepatocytes from humans (left) and rats (right) were incubated with paracetamol (Compound). Shown here are the acidification rate (red), respiratory rate (blue) and adhesion (green) of the primary hepatocytes. RM (running medium, medium without test substance), stand. rates (standardized rates), TX (0.2% Triton X-100 in RM, to kill the cells and used as a test for the sensors, showing that the recorded data is valid and represent no artefact).
The continuous process monitoring clearly shows the sequential influence on the cells' metabolism, which cannot be observed with end-point analysis methods. To investigate regeneration, only medium instead of medium + paracetamol is directed over the cells (from ca. 27 h, grey field). Whereas the human hepatocytes show complete regeneration, the acidification rate in the rat cells reaches only 85% and the oxygen rate only 70% of the original activity. The adhesion shows no regeneration at all, which indicates irreversible effects on the cells. While the human cells could be used for further tests, the rat hepatocytes have undergone irreversible damage due to paracetamol.
Applications in Stem Cell Research, GPCR Analysis and Apoptosis
Stem cell differentiation is characterized by a change in the ratio of mitochondrial respiration and glycolysis. By measuring oxygen consumption and extra cellular acidification, the sensor system permits predictions about the degree of stem cell differentiation, and thanks to the non-invasive and label-free analysis one is able to categorize the stem cells.
The sensor system can also be used to monitor receptor-specific activation from most GPCR's by measuring the impedance upon their stimulation. Finally, the sensor system monitors apoptosis-induced changes in cell shape in an integral and quantitative fashion with a high time resolution. Conversely, the sensor technology can also be used for proliferation assays.
Figure 2: The Bionas analyzing system allows one, two or six sensor chips to be analyzed in parallel.
Related news headlines to Bionas
Bionas make available its online cell monitoring system
21-Aug-2006 - Bionas has launched a product for in vitro profiling the metabolic activity of cells to understand cellular function that could prove highly useful in drug discovery and development.
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