Dielectric measurements on live biological materials under magnetic resonance conditions

In the course of work on the interactions of electric and magnetic fields with both living and dead biological materials, it was noticed that certain published dielectrophoretic yield curves for biological cells showed unexplained deviations in the region of 2 kHz. Dielectrophoretic measurements made at frequencies and magnetic fields which satisfied the nuclear magnetic resonance conditions showed sharply resonant features. Dielectric measurements showed small, but sharp, resonances most easily seen in the dielectric loss curves which had a bandwidth of the order of one Hertz and presented at the frequencies which satisfied the magnetic resonance conditions for the ambient magnetic field. Resonances were found corresponding to the frequencies for electron spin resonance and nuclear magnetic resonance for¹H,³¹P,²³Na,³⁷Cl and³⁹K. The onset of these resonances occurs at the value of the steady magnetic field strength so that one quantum of magnetic flux (2.07×10^?15wb) would link a single biological cell or pair of cells, approximately 1 G (100μT) in the case of a 5-μm yeast cell. The effects of these magnetic resonance conditions on the mean generation time ofE. coli and on the reaction of the enzyme lysozyme with the substrateM. lysodeikticus cells are also shown.     

Measurement of small mechanical vibrations of brain tissue exposed to extremely-low-frequency electric fields

Electromagnetic fields can interact with biological tissue both electrically and mechanically. This study investigated the mechanical interaction between brain tissue and an extremely-low-frequency (ELF) electric field by measuring the resultant vibrational amplitude. The exposure cell is a section of X-band waveguide that was modified by the addition of a center conductor to form a small TEM cell within the waveguide structure. The ELF signal is applied to the center conductor of the TEM cell. The applied ELF electric field generates an electrostrictive force on the surface of the brain tissue. This force causes the tissue to vibrate at a frequency equal to twice the frequency of the applied sinusoidal signal. An X-band signal is fed through the waveguide, scattered by the vibrating sample, and detected by a phase-sensitive receiver. Using a time-averaging spectrum analyzer, a vibration sensitivity of approximately 0.2 nmp-p can be achieved. The amplitude of the brain tissue vibrational response is constant for vibrational frequencies below 50 Hz; between 50 and 200 Hz resonant phenomena were observed; and above 200 Hz the amplitude fall-off is rapid.     

Lymphocyte electrotaxis in vitro and in vivo

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.     

The Influence of Electric Field and Confinement on Cell Motility

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.     

Alternating current electric field effects on neural stem cell viability and differentiation

Methods utilizing stem cells hold tremendous promise for tissue engineering applications; however, many issues must be worked out before these therapies can be routinely applied. Utilization of external cues for preimplantation expansion and differentiation offers a potentially viable approach to the use of stem cells in tissue engineering. The studies reported here focus on the response of murine neural stem cells encapsulated in alginate hydrogel beads to alternating current electric fields. Cell viability and differentiation was studied as a function of electric field magnitude and frequency. We applied fields of frequency (0.1–10) Hz, and found a marked peak in neural stem cell viability under oscillatory electric fields with a frequency of 1 Hz. We also found an enhanced propensity for astrocyte differentiation over neuronal differentiation in the 1 Hz cultures, as compared to the other field frequencies we studied. Published 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cellular spin resonance

Suspended cells can at times be seen to spin when in an ac electric field. The phenomenon is observed to be linked to cell colony age. We can now understand this in terms of the applied field acting synchronously upon a natural oscillating electric dipole associated with cell division. The dipole field strength thus estimated from spin drag agrees with the value earlier determined by the dielectrophoretic attraction (a non-uniform field effect on neutral bodies) of dividing cells for polarizable powders.Neither the source nor the role of the demonstrated rf electrical oscillations (about 10 000 Hz) of the dividing cells is as yet known. Whether the oscillations are necessary or incidental, is to be determined.     

Cellular spin resonance: Theory and experiment

Living and dead cells, as well as certain inanimate particles, can be made to spin in a rotating electric field, such as that produced by a four-pole electrode system. The theory for the effects and the experimental results for a number of cells are given. Yeast cells (Saccharomyces cerevisiae, dead, and as protoplasts;Schizosaccharomyces octospora); an alga (Chlorella pyrenoidosa); mouse myeloma cells; human Hela cells; and several model particles were studied. Their spectra of spin response are shown to agree with the theory presented. Interestingly, live cells spin in a contrafield direction, while dead ones spin co-field in the low-frequency range. The result indicate that this new technique of cellular spin resonance (CSR) will be useful in determining the effective dielectric constant of individual cells, for the direction and magnitude of the CSR is directly proportional to the effective dielectric constant, and the spin rate can readily and directly be determined over a wide frequency range.     

Cyclic AMP response in cells exposed to electric fields of different frequencies and intensities

The action on intracellular cyclic AMP (cAMP) of therapeutically used 4000-Hz electric fields was investigated and compared with 50-Hz data. Cultured mouse fibroblasts were exposed for 5 minutes to 4000-Hz sine wave internal electric fields between 3 mV/m and 30 V/m applied within culture medium. A statistically significant decrease in cellular cAMP concentration relative to unexposed cells was observed for fields higher than 10 mV/m. The drop in cAMP was most pronounced at lower field strengths (71 % of controls at 30 mV/m) and tended to disappear at higher field strengths. An increase of cAMP content was observed with 50-Hz electric fields, as was also the case when 4000-Hz fields were modulated with certain low frequencies.     

Experimental investigation on neural cell survival after dielectrophoretic trapping

Negative dielectrophoretic forces can effectively be used to trap cortical rat neurons. The creation of dielectrophoretic forces requires electric fields of high non-uniformity. High electric field strengths, however, can cause excessive membrane potentials by which cells may unrecoverably be changed or it may lead to cell death. In a previous study it was found that cells trapped at 3 Vtt/14 MHz did not change morphologically as compared to cells that were not exposed to the electric field. This study investigates the viability of fetal cortical rat neurons after being trapped by negative dielectrophoretic forces at frequencies up to 1 MHz. A planar quadrupole micro-electrode structure was used for the creation of a non-uniform electric field. The sinusoidal input signal was varied in amplitude (3 and 5 Vtt) and frequency (10 kHz-1 MHz). The results presented in this paper show that the viability of dielectrophoretically trapped postnatal cortical rat cells was greatly frequency dependent. To preserve viability frequencies above 100 kHz (at 3 Vtt) or 1 MHz (5 Vtt) must be used.     

Spinning response of yeast cells to rotating electric fields

The frequency-dependent rotation or spinning motion of yeast cells subjected to a fourpole rotating electric field was examined over a very wide frequency range (500 Hz to 500 MHz). In the lower frequency range (500 Hz – 700 KHz) the yeast cells were observed to spin in a direction counter to the applied field, with a small peak at about 600 Hz and a more pronounced one at 20 KHz. For frequencies above 700 KHz the spinning of the cells switched direction from counter-field to co-field, with a maximum in the rotation rate at about 70 MHz and a subpeak at 20 MHz. The rate was also observed to exhibit a square dependence on the magnitude of the applied rotating field.     

Dielectrophoretic manipulation of cells with spiral electrodes.

Electrokinetic responses of human breast cancer MDA-MB-231 cells were studied in suspensions of conductivities 18, 56, and 160 mS/m on a microelectrode array consisting of four parallel spiral electrode elements energized with phase-quadrature signals of frequencies between 100 Hz and 100 MHz. At low frequencies cells were levitated and transported toward or away from the center of the spiral array, whereas at high frequencies cells were trapped at electrode edges. The frequencies of transition between these characteristic cell behaviors increased with increasing suspension conductivity. Levitation heights and radial velocities were determined simultaneously for individual cells as a function of the applied field magnitude and frequency. Results were compared with theoretical predictions from generalized dielectrophoresis theory applied in conjunction with cell dielectric parameters and simulated electric field distributions corrected for electrode polarization effects. It was shown that the conventional and traveling-wave dielectrophoretic force components dominated cell levitation and radial motion, respectively. Both theoretical predictions and experimental data showed that the cell radial velocity was very sensitive to the field frequency when the in-phase component of the field-induced polarization was close to zero. Applications of spiral electrode arrays, including the isolation of cells of clinical relevance, are discussed.     

Electric Field-Induced Transmembrane Potential Depends on Cell Density and Organizatio

Electrochemotherapy is a novel technique to enhance the delivery of chemotherapeutic drugs into tumor cells. In this procedure, electric pulses are delivered to cancerous cells, which induce membrane permeabilization, to facilitate the passage of cytotoxic drugs through the cell membrane. This study examines how electric fields interact with and polarize a system of cells. Specifically, we consider how cell density and organization impact on induced cell transmembrane potential due to an external electric field. First, in an infinite volume of spherical cells, we examined how cell packing density impacts on induced transmembrane potential. With high cell density, we found that maximum induced transmembrane potential is suppressed and that the transmembrane potential distribution is altered. Second, we considered how orientation of cell sheets and strands, relative to the applied field, affects induced transmembrane potential. Cells that are parallel to the field direction suppress induced transmembrane potential, and those that lie perpendicular to the applied field potentiate its effect. Generally, we found that both cell density and cell organization are very important in determining the induced transmembrane potential resulting from an applied electric field.     

INFLUENCE OF LOW-FREQUENCY ELECTRIC FIELDS ON PROLIFERATION AND IL-8 RELEASE OF HL-60 CELLS

The influence of capacitively coupled extremely low-frequency (ELF) electric fields on proliferation and on interleukin (IL)-8 release of exponentially growing HL-60 cells was examined. The cell suspensions were treated with the field component of interferential current (IFC) using different exposure protocols. Modulation frequencies of 10 and 100 Hz were applied with field strengths between 0.075 and 11.54 V_pp/cm for 48 hr using a 5-min exposure time at every 3 hr. At a field strength of 1 V_pp/cm, the influence of the time between two exposure sessions was examined for different modulation frequencies. All exposure protocols applied have no effect on cell proliferation (p>0.05), but statistical significant reduction (p<0.05) of the IL-8 release at selected modulation frequencies and interval times could be observed.     

Extremely low frequency pulsed DC electric fields promote neutrophil extension, metabolic resonance and DNA damage when phase-matched with metabolic oscillators

Application of extremely low frequency pulsed DC electric fields that are frequency- and phase-matched with endogenous metabolic oscillations leads to greatly exaggerated neutrophil extension and metabolic resonance wherein oscillatory NAD(P)H amplitudes are increased. In the presence of a resonant field, migrating cell length grows from 10 to approximately 40 microm, as does the overall length of microfilament assemblies. In contrast, cells stop locomotion and become spherical when exposed to phase-mismatched fields. Although cellular effects were not found to be dependent on electrode type and buffer, they were sensitive to temporal constraints (phase and pulse length) and cell surface charge. We suggest an electromechanical coupling hypothesis wherein applied electric fields and cytoskeletal polymerization forces act together to overcome the surface/cortical tension of neutrophils, thus promoting net cytoskeletal assembly and heightened metabolic amplitudes. Metabolic resonance enhances reactive oxygen metabolic production by neutrophils. Furthermore, cellular DNA damage was observed after prolonged metabolic resonance using both single cell gel electrophoresis ('comet' assay) and 3'-OH DNA labeling using terminal deoxynucleotidyl transferase. These results provide insights into transmembrane signal processing and cell interactions with weak electric fields.     

Resonance electroconformational coupling: A proposed mechanism for energy and signal transductions by membrane proteins

Recent experiments show that membrane ATPases are capable of absorbing free energy from an applied oscillating electric field and converting it to chemical bond energy of ATP or chemical potential energy of concentration gradients. Presumably these enzymes would also respond to endogenous transmembrane electric fields of similar intensity and waveform. A mechanism is proposed in which energy coupling is achieved via Coulombic interaction of an electric field and the conformational equilibria of an ATPase. Analysis indicates that only an oscillating or fluctuating electric field can be used by an enzyme to drive a chemical reaction away from equilibrium.In vivo, the stationary transmembrane potential of a cell must be modulated to become locally oscillatory if it is to derive energy and signal transduction processes.     

Probing whole cell currents in high-frequency electrical fields: identification of thermal effects

An open-end coaxial probe is combined with a planar patch-clamp system to apply electric fields with GHz frequencies during conventional patch-clamp measurements. The combination of pulsed microwave irradiation and lock-in detection allows for the separation of fast and slow effects and hence facilitates the identification of thermal effects. The setup and the influence of radiation on the patch-clamp current are thoroughly characterized. For the independent optical verification of heating effects, a temperature microscopy technique is applied with high spatial, temporal and temperature resolution. It is shown that the effect of radiation at GHz frequencies on whole cell currents is predominantly thermal in nature in the case of RBL cells with an endogenous K(ir) 2.1 channel.     

Orientation of Schizosaccharomyces POMBE Nonliving Cells under Alternating Uniform and Nonuniform Electric Fields

When nonliving cells of Schizosaccharomyces pombe were subjected to the action of alternating uniform and nonuniform electric fields, two types of orientation were produced. The first one, with its longest axis parallel to the field lines, is similar to that obtained with living cells. The second, perpendicular to the direction of the field, is produced for relatively high frequencies and low conductivities; this probably takes place when the conductivities of the external and internal media (cell cytoplasm) become equal. A mixed cell population is produced in a discrete interval of the parameters used. Our results provide direct evidence that cell alignment does not depend on the physiological state of the cells.     

Orientation behavior of retinal photoreceptors in alternating electric fields

In alternating electric (AC) fields, particles experience polarizing effects that induce dipoles that orient elongated specimens either parallel or perpendicular to the field lines. In this work we studied the behavior of photoreceptor cells' rod outer segments (ROS) in AC fields of different frequencies. We showed that at low frequencies, ROS orient parallel to the field, whereas at higher frequencies they orient perpendicular to the field lines (in the frequency range from 100 Hz to 10 MHz). We found this behavior to be dependent on the physiological state of cells (due to modifications in their electrical properties). To simulate cell damage, the membrane conductivity was changed by treating the cell with gramicidin A, which resulted in a decrease of cytosol conductivity and, consequently, in a change of the orientation behavior of the treated cells. The change of cell orientation with cytosol conductivity is rather sharp, suggesting the potential of the method for accurate evaluation of the cell physiological status. We modeled the interaction between ROS and AC fields approximating the rod cell by a prolate spheroid with a very long axis. The internal compartment of the ellipsoid was considered to be filled with an inhomogeneous medium consisting of alternating layers of membrane and cytoplasm as media modeling the disks. This theoretical model proved to be in good agreement with the experimental results and enabled the derivation (by fitting with the experimental results) of the membrane and cytosol parameters for normal and damaged cells.     

Spatial properties of goldfish ganglion cells

We systematically classified goldfish ganglion cells according to their spatial summation properties using the same techniques and criteria used in cat and monkey research. Results show that goldfish ganglion cells can be classified as X-, Y-, or W-like based on their responses to contrast-reversal gratings. Like cat X cells, goldfish X-like cells display linear spatial summation. Goldfish Y-like cells, like cat Y cells, respond with frequency doubling at all spatial positions when the contrast-reversal grating consists of high spatial frequencies. There is also a third class of neurons, which is neither X- nor Y-like; many of these cells' properties are similar to those of the "not-X" cells found in the eel retina. Spatial filtering characteristics were obtained for each cell by drifting sinusoidal gratings of various spatial frequencies and contrasts across the receptive field of the cell at a constant temporal rate. The spatial tuning curves of the cell depend on the temporal parameters of the stimulus; at high drift rates, the tuning curves lose their low spatial frequency attenuation. To explore this phenomenon, temporal contrast response functions were derived from the cells' responses to a spatially uniform field whose luminance varied sinusoidally in time. These functions were obtained for the center, the surround, and the entire receptive field. The results suggest that differences in the cells' spatial filtering across stimulus drift rate are due to changes in the interaction of the center and surround mechanisms; at low temporal frequencies, the center and surround responses are out-of-phase and mutually antagonistic, but at higher temporal rates their responses are in-phase and their interaction actually enhances the cell's responsiveness.     

Cell culture dosimetry for low-frequency magnetic fields

Calculations of the current density and electric field distributions induced in cell cultures by an applied low-frequency magnetic field have assumed that the medium is uniform. This paper calculates these distributions for a more realistic, inhomogeneous, anisotropic model in which the cells are regarded as conducting squares surrounded by insulating membranes. Separate parameters are used to specify the resistivities of the cell interior, the cell membrane parallel to its surface, the cell membrane perpendicular to its surface, and the intercellular junction parallel to the membrane. The presence of gap junctions connecting the interiors of adjacent cells is also considered. For vertical applied magnetic fields, the induced currents and field distributions may deviate considerably from the homogeneous medium model if there is sufficiently tight binding of the cells to each other. The presence of gap junctions can produce relatively large transmembrane electric fields or intracellular current densities. These considerations are generally less important for horizontal applied fields. A simple microscopic model of the cell surface is also discussed. © 1996 Wiley-Liss, Inc.