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.