Parallel single-cell optical transit dielectrophoresis cytometer.

In this work, we present an optical transit DEP flow cytometer for parallel single-cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through the channel. These shadows are detected using a 256-pixel linear optical array detector. Massively parallel analysis is possible as each pixel of the detector can independently analyze the passing cells. A wide channel (∼18 mm) was employed to carry many particles simultaneously, and the system was capable of detecting the velocity of over 200 cells simultaneously. We have achieved analysis rates for 10 µm diameter polystyrene spheres response exceeding 250 per second. With appropriate calibration, this DEP cytometer can quantitatively measure the dielectric response. The dielectric response (Clausius-Mossotti factor) of viable CHO cells was measured over the frequency range of 100 kHz to 6 MHz, and the obtained response matches the previously measured values by our group. The DEP cytometer uses simple modular components to achieve high throughput label-free single-cell dielectric analysis and can begin analyzing particles within 10 s after starting to pump the sample into the channel.

Cytoplasmic conductivity as a marker for bioprocess monitoring: Study of Chinese hamster ovary cells under nutrient deprivation and reintroduction.

The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na+ /K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48-hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na+ /K+ ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient-deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.

Progression of change in membrane capacitance and cytoplasm conductivity of cells during controlled starvation using dual-frequency DEP cytometry.

The dielectric properties of cells are directly related to their morphological and physiological properties and can be used to monitor their status when exposed to stress conditions. In this work, dual-frequency dielectrophoresis (DEP) cytometry was employed to measure changes in the membrane capacitance and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells during the progression of starvation-induced apoptosis. Our dual-frequency DEP cytometer enables simultaneous measurement of multiple dielectric properties of single cells and identification of their state (viable or apoptotic) within a heterogeneous sample. We employed one frequency to determine each cell's viability state and the other frequency to characterize the change in membrane capacitance or cytoplasm conductivity. Cells were starved by incubation in a medium lacking glucose and glutamine and monitored every 12 h over a 64 h period. Our results showed a subpopulation of early apoptotic cells emerged after 40 h in the starvation medium, which rapidly increased during the next 12 h. After 52 h, a complete transition from viable to apoptotic state was observed. Analyzing the subpopulation of viable cells over the first 52 h showed that the membrane capacitance gradually declined from an initial value of 2.0 to 1.2 µF/cm2, and was 0.9 µF/cm2 for apoptotic cells. The cytoplasm conductivity of viable cells initially remained constant and then declined from 0.40 to 0.27 S/m after 40 h, coinciding with onset of apoptotic processes. A dramatic decrease in cytoplasm conductivity from 0.27 to 0.07 S/m was observed after 52 h, corresponding to apoptotic cells. As membrane capacitance is related to membrane morphology and cytoplasm conductivity is related to intracellular ion concentrations, the results indicate that during controlled starvation the cell membrane smooths gradually whereas intracellular ion concentrations are initially maintained near homeostatic levels until a later dramatic decline occurs.

Quantitative Model for Ion Transport and Cytoplasm Conductivity of Chinese Hamster Ovary Cells.

In mammalian cells cytoplasm ion concentrations and hence cytoplasm conductivity is an important indicator of their physiological state. Changes in the cytoplasm conductivity has been associated with physiological changes such as progression of cancer and apoptosis. In this work, a model that predicts the effects of physiological changes in ion transport on the cytoplasm conductivity of Chinese hamster ovary (CHO) cells is demonstrated. We determined CHO-specific model parameters, Na+/K+ ATPase pumps and ion channels densities, using a flux assay approach. The obtained sodium (PNa), potassium (PK) and chloride (PCl) permeability and Na+/K+ ATPase pump density were estimated to be 5.6 × 10-8 cm/s, 5.6 × 10-8 cm/s, 3.2 × 10-7 cm/s and 2.56 × 10-11 mol/cm2, respectively. The model was tested by comparing the model predictions with the experimentally determined temporal changes in the cytoplasm conductivity of Na+/K+ ATPase pump inhibited CHO cells. Cells' Na+/K+ ATPase pumps were inhibited using 5 mM Ouabain and the temporal behavior of their cytoplasm conductivity was measured using dielectrophoresis cytometry. The measured results are in close agreement with the model-calculated values. This model will provide insight on the effects of processes such as apoptosis or external media ion concentration on the cytoplasm conductivity of mammalian cells.

Single cell dielectrophoresis study of apoptosis progression induced by controlled starvation.

Nutrient depletion in fed-batch cultures and at the end of batch cultures is among the main causes of stress on cells and a trigger of apoptosis. In this study, we investigated changes in the cytoplasm conductivity of Chinese hamster ovary (CHO) cells under controlled starvation. Employing a single-cell dielectrophoresis (DEP) cytometer, we measured the DEP response of CHO cells incubated in a medium without glucose and glutamine over a 48-h period. Using the measured data in conjunction with numerical simulations, we determined the cytoplasm conductivity of viable and apoptotic cell subpopulations. The results show that a small subpopulation of apoptotic cells emerges after 24 to 36 h of starvation and increases rapidly over a short period of time, <12 h. The apoptotic cells have a dramatically lower cytoplasm conductivity, ∼0.05 S/m, than viable cells, ∼0.45 S/m. Viability of starvation cultures was measured by fluorescent cytometry, DEP cytometry, and trypan blue exclusion assays. DEP, Annexin V, caspase-8, and 7-AAD assays show a similar decline in viability after 36 h of starvation and indicate a very low viability after 48 h. Trypan blue exclusion assay fails to detect early-stage viability decline and estimates a much higher viability after 48 h.

The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess.

To ensure maximum productivity of recombinant proteins it is desirable to prolong cell viability during a mammalian cell bioprocess, and therefore important to carefully monitor cell density and viability. In this study, five different and independent methods of monitoring were applied to Chinese hamster ovary (CHO) cells grown in a batch culture in a controlled bioreactor to determine cell density and/or cell viability. They included: a particle counter, trypan blue exclusion (Cedex), an in situ bulk capacitance probe, an off-line fluorescent flow cytometer, and a prototype dielectrophoretic (DEP) cytometer. These various techniques gave similar values during the exponential growth phase. However, beyond the exponential growth phase the viability measurements diverged. Fluorescent flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis: the cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay showed higher estimates of viable cell density and viability compared to the capacitance probe or the DEP cytometer. The DEP cytometer measures the dielectric properties of individual cells and identified at least two populations of cells, each with a distinct polarizability. As verified by comparison with the Nexin assay, one population was associated with viable (non-apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline stages there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells between different dielectric sub-populations, rather than assuming a homogeneous dielectric population. This shows that bulk dielectric probes are sensitive to the early apoptotic changes in cells. DEP cytometry offers a novel and unique technology for analyzing and characterizing mammalian cells based on their dielectric properties, and suggests a potential application of the device as a low-cost, label-free, electronic monitor of physiological changes in cells.

Differential electronic detector to monitor apoptosis using dielectrophoresis-induced translation of flowing cells (dielectrophoresis cytometry).

The instrument described here is an all-electronic dielectrophoresis (DEP) cytometer sensitive to changes in polarizability of single cells. The important novel feature of this work is the differential electrode array that allows independent detection and actuation of single cells within a short section ([Formula: see text]) of the microfluidic channel. DEP actuation modifies the altitude of the cells flowing between two altitude detection sites in proportion to cell polarizability; changes in altitude smaller than 0.25 µm can be detected electronically. Analysis of individual experimental signatures allows us to make a simple connection between the Clausius-Mossotti factor (CMF) and the amount of vertical cell deflection during actuation. This results in an all-electronic, label-free differential detector that monitors changes in physiological properties of the living cells and can be fully automated and miniaturized in order to be used in various online and offline probes and point-of-care medical applications. High sensitivity of the DEP cytometer facilitates observations of delicate changes in cell polarization that occur at the onset of apoptosis. We illustrate the application of this concept on a population of Chinese hamster ovary (CHO) cells that were followed in their rapid transition from a healthy viable to an early apoptotic state. DEP cytometer viability estimates closely match an Annexin V assay (an early apoptosis marker) on the same population of cells.

Electronic detection of dielectrophoretic forces exerted on particles flowing over interdigitated electrodes.

Dielectric particles flowing through a microfluidic channel over a set of coplanar electrodes can be simultaneously capacitively detected and dielectrophoretically (DEP) actuated when the high (1.45 GHz) and low (100 kHz-20 MHz) frequency electromagnetic fields are concurrently applied through the same set of electrodes. Assuming a simple model in which the only forces acting upon the particles are apparent gravity, hydrodynamic lift, DEP force, and fluid drag, actuated particle trajectories can be obtained as numerical solutions of the equations of motion. Numerically calculated changes of particle elevations resulting from the actuation simulated in this way agree with the corresponding elevation changes estimated from the electronic signatures generated by the experimentally actuated particles. This verifies the model and confirms the correlation between the DEP force and the electronic signature profile. It follows that the electronic signatures can be used to quantify the actuation that the dielectric particle experiences as it traverses the electrode region. Using this principle, particles with different dielectric properties can be effectively identified based exclusively on their signature profile. This approach was used to differentiate viable from non-viable yeast cells (Saccharomyces cerevisiae).