Classifying contaminated cell cultures using time series features.

We examine the use of time series data, derived from Electric Cell-substrate Impedance Sensing (ECIS), to differentiate between standard mammalian cell cultures and those infected with a mycoplasma organism. With the goal of easy visualization and interpretation, we perform low-dimensional feature-based classification, extracting application-relevant features from the ECIS time courses. We can achieve very high classification accuracy using only two features, which depend on the cell line under examination. Initial results also show the existence of experimental variation between plates and suggest types of features that may prove more robust to such variation. Our paper is the first to perform a broad examination of ECIS time course features in the context of detecting contamination; to combine different types of features to achieve classification accuracy while preserving interpretability; and to describe and suggest possibilities for ameliorating plate-to-plate variation.

Automated measurement of transepithelial electrical resistance (TEER) in 96-well transwells using ECIS TEER96: Single and multiple time point assessments.

Transepithelial electrical resistance (TEER) is a widely used technique for quantifying the permeability of epithelial and endothelial cell layers. However, traditional methods of measuring TEER are limited to single timepoint measurements and can subject cells to an altered environment during the measurement. Here, we assessed the validity of TEER measurements by the ECIS TEER96 device, which is designed to take continuous TEER measurements of a cell culture system in a standard laboratory incubator. We found that the instrument accurately measures TEER across TEER values ranging from 10 to 2050 Ω*cm2 and is more accurate than the manual epithelial voltohmmeter electrode at high TEER values. Furthermore, the high-resolution measurements provided by the device allowed for a unique insight into the mechanisms and kinetics of cells in vitro. To demonstrate the continuous measurement capability of the device, we tracked the formation of an MDCKI cell monolayer until TEER plateaued. Furthermore, we treated Caco-2 monolayers with different concentrations of DMSO and the antimicrobial and surfactant compound benzethonium chloride to measure disruptions to barrier integrity. Treatment of both compounds resulted in concentration-dependent loss of barrier integrity. Our results suggest that the ECIS TEER96 device is a reliable and convenient option for measuring TEER in cell cultures and can provide valuable insights into the behavior of cells in vitro. This technology will be especially useful for increasing throughput of drug permeability assays, inflammation studies, and gaining better understanding of disease states in a cell culture system.

Rapid assay of stem cell functionality and potency using electric cell-substrate impedance sensing.

Regenerative medicine studies using autologous bone marrow mononuclear cells (BM-MNCs) have shown improved clinical outcomes that correlate to in vitro BM-MNC invasive capacity. The current Boyden-chamber assay for testing invasive capacity is labor-intensive, provides only a single time point, and takes 36 hours to collect data and results, which is not practical from a clinical cell delivery perspective. To develop a rapid, sensitive and reproducible invasion assay, we employed Electric Cell-substrate Impedance Sensing (ECIS) technology. Chemokine-directed BM-MNC cell invasion across a Matrigel-coated Transwell filter was measurable within minutes using the ECIS system we developed. This ECIS-Transwell chamber system provides a rapid and sensitive test of stem and progenitor cell invasive capacity for evaluation of stem cell functionality to provide timely clinical data for selection of patients likely to realize clinical benefit in regenerative medicine treatments. This device could also supply robust unambiguous, reproducible and cost effective data as a potency assay for cell product release and regulatory strategies.

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations.

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 µm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).

Electrical wound-healing assay for cells in vitro.

Confluent cell monolayers in tissue culture are fragile and can easily be mechanically disrupted, often leaving an area devoid of cells. This opening in the cell sheet is then repopulated, because the cells on the fringe of the damage, which are no longer contact-inhibited, move into the available space. This mechanical disruption is often done deliberately in a "wound-healing" assay as a means to assess the migration of the cells. In such assays, a scrape is made in the cell layer followed by microscopy to monitor the advance of the cells into the wound. We have found that these types of assays can also be accomplished electrically. In this approach, cells growing on small electrodes and monitored by using electric cell-substrate impedance sensing are subjected to currents, resulting in severe electroporation and subsequent cell death. After this invasive treatment, the electrode's impedance is again monitored to chart the migration and ultimate healing of the wound. We report here that this procedure to study cell behavior is both highly reproducible, quantitative, and provides data similar to that acquired with traditional measurements.

Real-time impedance assay to follow the invasive activities of metastatic cells in culture.

Here we present research detecting the invasive activities of metastatic cells in vitro using electric cell-substrate impedance sensing (ECIS). The assay is based on previous microscopic observations, where metastatic cells added over established endothelial cell layers were observed to attach to and invade the cell layer. Human umbilical vein endothelial cells (HUVECs) werefirst grown to confluence on small gold electrodes. The impedance of these electrodes was followed after the addition of suspensions of different sublines of the Dunning murine prostatic adenocarcinoma series (G, AT1, AT2, AT3, ML, and MLL). For highly metastatic sublines, within an hour after being challenged, the impedance of the confluent HUVEC layer was substantially reduced. The effect of the weakly metastatic sublines was less pronounced, and the extent and the rate of this drop in impedance could be correlated with the metastatic potential of each of six sublines tested. The real-time assay is effective in both normal and low (1%) serum concentrations, and the detected activity requires the presence of viable transformed cells. In addition to the murine cell lines, similar behavior was observed using four established human prostatic cancer lines (DU145, PC3, TSU, and PPC1). These results suggest that this ECIS-based assay might be used with primary human cultures to establish the metastatic abilities of cells isolated from biopsies.

Recovery of adherent cells after in situ electroporation monitored electrically.

Here we describe various experiments that address the efficiency of loading extracellular probes into the cytoplasm of adherent mammalian cells (normal rat kidney, Madin-Darby canine kidney, and African green monkey) by means of in situ electroporation. Subsequent cell recovery from the electroporation pulse was monitored electrically in real time for each condition. In this study, small, gold-film electrodes (5 x 10(-4) cm2) are used as culture substrates and at the same time as an electrode for both the application of the electroporating voltage pulse and the noninvasive electrical monitoring of cell recovery, using a technique referred to as ECIS. Electroporation has been performed by using ac sinusoidal voltage pulses of varying frequency, amplitude, and duration. Permeabilization and re-closure of the plasma membrane were evaluated by the uptake of the fluorescence probe, Lucifer Yellow, from the extracellularfluid. With the experimental setup described here, efficient electroporation was achieved with voltages less than 5 V. Using ECIS, we followed the morphological response of the cells to the electricfield-induced membrane permeabilization. For optimized electroporation conditions, cell recovery was completed in less than 1 h. The introduction of membrane-impermeable substances by electroporation and in situ monitoring of the cellular response mayfind many applications in cell biology.