Elham Salimi

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. Biotechnol. Bioeng. 2013;110: 2902–2914.

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).

Torque-mixing magnetic resonance spectroscopy

Mechanically detected spin resonances The interaction of spins in a sample with a magnetic field can generate forces that can be sensed with cantilever probes. Losby et al. measured the resonance signals at room temperature with a micromechanical torque magnetometer. The difference between two applied radio-frequency signals corresponded to the mechanical frequency of the resonator. This approach revealed the vortex core dynamics of the ferri-toferro–magnetic transition in a micrometer-sized yttrium-iron-garnet single-crystal disk. Science, this issue p. 798 Electronic spin resonances of a magnetic single crystal are measured with a mechanical torque sensor. A universal, torque-mixing method for magnetic resonance spectroscopy is presented. In analogy to resonance detection by magnetic induction, the transverse component of a precessing dipole moment can be measured in sensitive broadband spectroscopy, here using a resonant mechanical torque sensor. Unlike induction, the torque amplitude allows equilibrium magnetic properties to be monitored simultaneously with the spin dynamics. Comprehensive electron spin resonance spectra of a single-crystal, mesoscopic yttrium iron garnet disk at room temperature reveal assisted switching between magnetization states and mode-dependent spin resonance interactions with nanoscale surface imperfections. The rich detail allows analysis of even complex three-dimensional spin textures. The flexibility of microelectromechanical and optomechanical devices combined with broad generality and capabilities of torque-mixing magnetic resonance spectroscopy offers great opportunities for development of integrated devices.

Electroporation and dielectrophoresis of single cells using a microfluidic system employing a microwave interferometric sensor

We describe a microfluidic device integrated with a microwave interferometric sensor that is able to simultaneously electroporate and measure the dielectrophoresis (DEP) response of single biological cells. The system can measure changes in dielectric properties of a cell permeabilized using a high-intensity pulsed electric field (PEF) from a few seconds after exposure to the pulses. It provides a mechanism for investigating time-dependent changes in the cell membrane and ion flux. Using this device, experiments performed on single Chinese hamster ovary (CHO) cells exposed to microsecond pulsed electric fields show significant changes in their DEP response immediately after exposure.

Multi-Frequency DEP Cytometer Employing a Microwave Sensor for Dielectric Analysis of Single Cells

We present a microfluidic device for in-flow dielectric characterization of single biological cells. The dielectric spectrum is obtained by measuring the multiple-frequency dielectrophoresis (DEP) response of individual cells as they travel over an array of sensing and actuating electrodes. The DEP induced translation of each cell is detected by measuring the differential impedance of the array using a microwave interferometer, which is capable of sub-attofarad sensitivity, and is coupled to the sensing electrodes. The DEP response of a cell at multiple frequencies in the beta-dispersion region is chosen to discern particular cell dielectric properties as it travels along the array-such as cytoplasm conductivity and membrane capacitance. The Clausius-Mossotti factor of the cell is determined from the measured response signal in conjunction with numerical simulation of its trajectory. The approach is validated through measuring polystyrene microspheres. The DEP response of Chinese hamster ovary cells using two simultaneous frequencies is demonstrated.

Multi-frequency DEP cytometer employing a microwave interferometer for the dielectric analysis of micro-particles

We present a multi-frequency dielectrophoresis (DEP) based microfluidic device for characterizing the complex dielectric properties of single micron-sized particles while in flow. The device employs a multi-electrode transmission line sensor coupled to a microwave-interferometer, capable of sub-attofarad sensitivity, for detecting the DEP-induced translation of the particle under study. DEP actuation of the particle at different frequencies - which is related to its dielectric response - is sensed as it travels along the sensor. Characterization of the dielectric response of polystyrene micro-spheres using two frequencies is demonstrated.

Dielectrophoresis study of temporal change in internal conductivity of single CHO cells after electroporation by pulsed electric fields

Applying sufficiently strong pulsed electric fields to a cell can permeabilize the membrane and subsequently affect its dielectric properties. In this study, we employ a microfluidic dielectrophoresis cytometry technique to simultaneously electroporate and measure the time-dependent dielectric response of single Chinese hamster ovary cells. Using experimental measurements along with numerical simulations, we present quantitative results for the changes in the cytoplasm conductivity of single cells within seconds after exposure to 100 μs duration pulsed electric fields with various intensities. It is shown that, for electroporation in a medium with conductivity lower than that of the cells cytoplasm, the internal conductivity of the cell decreases after the electroporation on a time scale of seconds and stronger pulses cause a larger and more rapid decrease. We also observe that, after the electroporation, the cells internal conductivity is constrained to a threshold. This implies that the cell prevents some of the ions in its cytoplasm from diffusing through the created pores to the external medium. The temporal change in the dielectric response of each individual cell is continuously monitored over minutes after exposure to pulsed electric fields. A time constant associated with the cells internal conductivity change is observed, which ranges from seconds to tens of seconds depending on the applied pulse intensity. This experimental observation supports the results of numerical models reported in the literature.

In-flow dielectric characterization of single biological cells using a wideband DEP cytometer

We describe a microfluidic dielectrophoresis (DEP) cytometer that is able to measure the dielectric properties of single biological cells while in flow over the 100 kHz - 400 MHz frequency range. The device provides both the sign and magnitude of the Clausius-Mossotti factor over the entire beta-dispersion region. A microwave interferometer is used to measure the DEP induced translation of individual cells as they flow over a multi-electrode sensing array. The DEP response of polystyrene microspheres is used to verify and calibrate the device. The spectral response of Chinese hamster ovary cells (CHO) is measured and the corresponding cross-over frequencies are determined.

Dielectric properties of CHO cells obtained using a microwave interferometer based dielectrophoresis cytometer

Studying biological phenomena at the cellular level allows understanding the basics of cellular function and the mechanism of their interactions with internal or external stimuli. Among available single cell analysis techniques such as, fluorescent labeling, magnetic labeling, mechanical, and electrical approaches, electrical ones are of special interest due to their minimum influence on the natural state of cells. Dielectrophoresis (DEP) cytometry- translation of cells in a non-uniform electric field- is an electrical based technique previously employed for differentiation and characterization of cells (R. Pethig, Biomicrofluidics, 2010). In this work we employ a microwave based DEP cytometry technique that we have developed to obtain dielectric parameters of Chinese Hamster Ovary (CHO) cells, a cell line with prominent pharmaceutical applications for production of therapeutic proteins. Cell electrical parameters are not well established. We present a model for CHO using a double shell model comprising the cell membrane, cytoplasm, nuclear envelope, and nucleus.