BackgroundDistinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.Methodology/Principal FindingsWe used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.Conclusions/SignificanceWe show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Dielectrophoresis (DEP) offers many advantages over conventional cell assays such as flow cytometry and patch clamp techniques for assessing cell electrophysiology as a marker for cancer studies and drug interaction assessment. However, despite the advantages offered by DEP analysis, uptake has been low, remaining largely in the academic arena, due to the process of analysis being time-consuming, laborious, and ultimately allowing only serial analysis on small numbers of cells. In this paper we describe a new method of performing DEP analysis based on laminate manufacturing methods. These use a three-dimensional “well” structure, similar in size and pitch to conventional microtiter well plates, but offer electrodes along the inner surface to allow easy measurement of cell properties through the whole population. The result can then be determined rapidly using a conventional well-plate reader. The nature of the device means that many electrodes, each containing a separate sample, can be tested in parallel, while the mode of observation means that analysis can be combined with simultaneous measurement of conventional fluorimetric well-based assays. Here we benchmark the device against standard DEP assays, then show how such a device can be used to (a) rapidly determine the effects both of ion channel blockers on cancer cells and antibiotics on bacteria and (b) determine the properties of multiple subpopulations of cells within a well simultaneously.

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V.

Dielectrophoretic and electrohydrodynamic forces have been demonstrated in the literature to cause the movement of particles across the surface of planar electrodes when exposed to low-frequency (approx. 1kHz) electric fields. In this paper we describe the development of this phenomenon for the collection of particles, covering a range of sizes, out of a liquid and focusing them at the centercentre of a novel electrode consisting of large interlocking circles. The volume of analyte across which this effect is observed is significantly larger than has been reported for conventional dielectrophoretic arrays. By altering the experimental conditions, particles can either be collected or cycled across the surface and then removed. This technique offers great scope for the enhancement of surface-based detection methods.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2 > 0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells – from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2–3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

Induced particle motion by dielectrophoresis has been assessed on a microelectrode system consisting of two facing planes, one with circular apertures which we have termed "dots". Dot diameters ranging between 150µm -500µm have been used to show positive and negative dielectrophoresis, and have shown differences in particle velocities which is also influenced by the concentration of the suspended particles in the system. Simulations of the electric field morphology within the system showed a characteristic dome-shape distribution over the centre of the dot for smaller diameters which becomes distorted as the diameter increases. The design of the microelectrode geometry has effective spatial electric field penetrating capabilities over planar dielectrophoretic electrode geometries, with the potential for novel on chip processes such as particle characterisation which is described in this paper.