Proposal and performance evaluation of a new parallel plate continuous cell separation device using dielectrophoresis.

Along with the rapid development of cellular biological research in recent years, there has been an urgent need for a high-speed, high-precision method of separating target cells from a highly heterogeneous cell population. Among the various cell separation technologies proposed so far, dielectrophoresis (DEP)-based approaches have shown particular promise because they are noninvasive to cells. We have developed a new DEP-based device to separate large numbers of live and dead cells of the human mammary cell line MCF10A. In this study, we validated the separation performance of this device. The results showed the successful separation of a higher percentage of cells than in previous studies, with a separation efficiency higher than 90%. In the past, there have been no confirmed cases in which a separation rate of over 90% and high-speed processing of a large number of cells were simultaneously achieved. It was shown that the proposed device can process large numbers of cells at high speed and with high accuracy.

Low-energy shock waves evoke intracellular Ca2+ increases independently of sonoporation.

Low-energy shock waves (LESWs) accelerate the healing of a broad range of tissue injuries, including angiogenesis and bone fractures. In cells, LESW irradiations enhance gene expression and protein synthesis. One probable mechanism underlying the enhancements is mechanosensing. Shock waves also can induce sonoporation. Thus, sonoporation is another probable mechanism underlying the enhancements. It remains elusive whether LESWs require sonoporation to evoke cellular responses. An intracellular Ca2+ increase was evoked with LESW irradiations in endothelial cells. The minimum acoustic energy required for sufficient evocation was 1.7 µJ/mm2. With the same acoustic energy, sonoporation, by which calcein and propidium iodide would become permeated, was not observed. It was found that intracellular Ca2+ increases evoked by LESW irradiations do not require sonoporation. In the intracellular Ca2+ increase, actin cytoskeletons and stretch-activated Ca2+ channels were involved; however, microtubules were not. In addition, with Ca2+ influx through the Ca2+ channels, the Ca2+ release through the PLC-IP3-IP3R cascade contributed to the intracellular Ca2+ increase. These results demonstrate that LESW irradiations can evoke cellular responses independently of sonoporation. Rather, LESW irradiations evoke cellular responses through mechanosensing.

Insulator-based dielectrophoresis combined with the isomotive AC electric field and applied to single cell analysis.

We developed an insulator-based dielectrophoretic (iDEP) creek-gap device that enables the isomotive movement of cells and that is suitable for determining their DEP properties. In the iDEP creek-gap device, a pair of planar insulators forming a single fan-shaped channel allows the induction of the isomotive iDEP force on cells. Hence, the cells' behavior is characterized by straight motion at constant velocity in the longitudinal direction of the channel. Operation of the device was demonstrated using human breast epithelial cells (MCF10A) by applying an AC voltage of Vpp = 34 V peak-to-peak and frequencies of 200 kHz and 50 MHz to the device. Subsequently, the magnitude of DEP forces and the real part of the ClausiusMossotti (CM) factor, Re(ß), were deduced from the measured cell velocity. The values of Re(ß) were 0.14 ± 0.01 for the frequency of 200 kHz and -0.12 ± 0.01 for 50 MHz. These results demonstrated that the DEP properties of the cells could be extracted over a wide field frequency range. Therefore, the proposed iDEP creek-gap device was found to be applicable to cell analysis.

Analysis of the dielectrophoretic properties of cells using the isomotive AC electric field.

Various microfluidic devices utilizing the principle of dielectrophoresis (DEP) have been developed to separate, concentrate, and characterize biological cells; however, their performance is still limited by a lack of quantitative characterization. We addressed this limitation by employing a method capable of accurately quantifying a cell's response to an imposed field gradient. In this study, a simple method using a newly designed Creek-gap electrode was proposed, and the electrokinetic behavior of cells was characterized by DEP velocimetry under the exposure of an approximately constant gradient of electric field square established along the gap of the electrodes. Together with the numerical prediction of the electric field based on three-dimensional electric field analysis, the magnitude of DEP forces and the real part of the Clausius-Mossotti factor of cells were deduced from their movement. Results demonstrated that the proposed method was applicable to the determination of the dielectrophoretic properties of cells.

High-throughput separation of cells by dielectrophoresis enhanced with 3D gradient AC electric field.

We propose a novel, high-performance dielectrophoretic (DEP) cell-separation flow chamber with a parallel-plate channel geometry. The flow chamber, consisting of a planar electrode on the top and an interdigitated-pair electrode array at the bottom, was developed to facilitate the separation of cells by creating a nonuniform AC electric field throughout the volume of the flow chamber. The operation and performance of the device were evaluated using live and dead human epithermal breast (MCF10A) cells. The separation dynamics of the cell suspension in the flow chamber was also investigated by numerically simulating the trajectories of individual cells. A theoretical model to describe the dynamic cell behavior under the action of DEP, including dipole-dipole interparticle, viscous, and gravitational forces, was developed. The results demonstrated that the live cells traveling through the flow chamber congregated into sites where the electric field gradient was minimal, in the middle of the flow stream slightly above the centerlines of the grounded electrodes at the bottom. Meanwhile, the dead cells were trapped on the edges of the high-voltage electrodes at the bottom. Cells were thus successfully separated with a remarkably high separation ratio (∼98%) at the appropriately tuned field frequency and applied voltage. The numerically predicted behavior and spatial distribution of the cells during separation also showed good agreement with those observed experimentally.

Modeling and simulation of dielectrophoretic collective dynamics in a suspension of polarizable particles under the action of a gradient AC electric field.

When a suspension of polarizable particles is subjected to a gradient AC electric field, the particles exhibit collective motion due to an interaction between the dipole induced in the particles and the spatial gradient of the electric field; this is known as dielectrophoresis. In the present study, the collective dynamics of suspended particles in a parallel-plate electric chamber was investigated by simulating numerically the trajectories of individual particles under the action of combined dielectrophoretic and dipole-dipole interparticle forces. The particles were transported by the dielectrophoretic forces toward the grounded electrodes. Before long, when the particles approached the site of the minimum field strength, attractive/repulsive interparticle forces became dominant and acted among the particles attempting to form a column-like cluster, having the particles distribution in concentric circles in its cross-section, in line with the centerline of the grounded electrodes. Our results also well reproduced the transient particle aggregation that was observed experimentally.

Enhancement of continuous-flow separation of viable/nonviable yeast cells using a nonuniform alternating current electric field with complex spatial distribution.

The variability in cell response to AC electric fields is selective enough to separate not only the cell types but also the activation states of similar cells. In this work, we use dielectrophoresis (DEP), which exploits the differences in the dielectric properties of cells, to separate nonviable and viable cells. A parallel-plate DEP device consisting of a bottom face with an array of micro-fabricated interdigitated electrodes and a top face with a plane electrode was proposed to facilitate the separation of cells by creating a nonuniform electric field throughout the flow channel. The operation and performance of the device were evaluated using live and dead yeast cells as model biological particles. Further, numerical simulations were conducted for the cell suspensions flowing in a channel with a nonuniform AC electric field, modeled on the basis of the equation of motion of particles, to characterize the separation efficiency by changing the frequency of applied AC voltage. Results demonstrated that dead cells traveling through the channel were focused onto a site around the minimum electric field gradient in the middle of the flow stream, while live cells were trapped on the bottom face. Cells were thus successfully separated under the appropriately tuned frequency of 1 MHz. Predictions showed good agreement with the observation. The proposed DEP device provides a new approach to, for instance, hematological analysis or the separation of different cancer cells for application in circulating tumor cell identification.

Visible-Light-Induced Morphological Changes of Giant Vesicles by Photoisomerization of a Ruthenium Aqua Complex.

Visible- and red-light responsive vesicles were prepared by incorporating a ruthenium aqua complex having two alkyl chains on tridentate and asymmetrical bidentate ligands (proximal-2: [Ru(C10 tpy)(C10 pyqu)OH2 ](2+) , C10 tpy=4'-decyloxy-2,2';6',2"-terpyridine, C10 pyqu=2-[2'-(6'-decyloxy)-pyridyl]quinoline). The ruthenium complex of proximal-2 with closed alkyl chain geometry and a cylinder-like molecular shape exhibited photoisomerization to distal-2 with an open alkyl chain geometry and a cone-like shape, both in an aqueous solution and in vesicle dispersions. We observed that light irradiation of giant vesicles containing proximal-2 induced diverse morphological changes.

Numerical simulation of dielectrophoretic separation of live/dead cells using a three-dimensional nonuniform AC electric field in micro-fabricated devices.

BACKGROUND: The analysis of cell separation has many important biological and medical applications. Dielectrophoresis (DEP) is one of the most effective and widely used techniques for separating and identifying biological species. OBJECTIVE: In the present study, a DEP flow channel, a device that exploits the differences in the dielectric properties of cells in cell separation, was numerically simulated and its cell-separation performance examined. METHODS: The samples of cells used in the simulation were modeled as human leukocyte (B cell) live and dead cells. The cell-separation analysis was carried out for a flow channel equipped with a planar electrode on the channel's top face and a pair of interdigitated counter electrodes on the bottom. This yielded a three-dimensional (3D) nonuniform AC electric field in the entire space of the flow channel. RESULTS: To investigate the optimal separation conditions for mixtures of live and dead cells, the strength of the applied electric field was varied. With appropriately selected conditions, the device was predicted to be very effective at separating dead cells from live cells. CONCLUSIONS: The major advantage of the proposed method is that a large volume of sample can be processed rapidly because of a large spacing of the channel height.

Dielectric aggregation kinetics of cells in a uniform AC electric field.

BACKGROUND: Cell manipulation and separation technologies have potential biological and medical applications, including advanced clinical protocols such as tissue engineering. OBJECTIVE: An aggregation model was developed for a human carcinoma (HeLa) cell suspension exposed to a uniform AC electric field, in order to explore the field-induced structure formation and kinetics of cell aggregates. METHODS: The momentum equations of cells under the action of the dipole-dipole interaction were solved theoretically and the total time required to form linear string-like cluster was derived. The results were compared with those of a numerical simulation. Experiments using HeLa cells were also performed for comparison. RESULTS: The total time required to form linear string-like clusters was derived from a simple theoretical model of the cell cluster kinetics. The growth rates of the average string length of cell aggregates showed good agreement with those of the numerical simulation. In the experiment, cells were found to form massive clusters on the bottom of a chamber. The results imply that the string-like cluster grows rapidly by longitudinal attraction when the electric field is first applied and that this process slows at later times and is replaced by lateral coagulation of short strings. CONCLUSIONS: The findings presented here are expected to enable design of methods for the organization of three-dimensional (3D) cellular structures without the use of micro-fabricated substrates, such as 3D biopolymer scaffolds, to manipulate cells into spatial arrangement.

Contactless cell trapping by the use of a uniform AC electric field.

The AC electric field-driven manipulation of suspended polarizable particles has become a major technique in micro- and nano-devices. In the present study, suspensions of cultured HeLa cells in isotonic solution were used to explore the mechanisms underlying the suspension behaviors during exposure to a uniform AC electric field of strength E(rms)=1.67×10(4) V/m at frequency 1 kHz. Molecular dynamics (MD) simulations based on the Langevin equation of particle kinetics were performed to elucidate the corresponding problem. A theoretical model to compute the trajectories of individual cells under the action of electro-mechanical, viscous and gravitational forces in the suspending medium was newly developed. Numerical computations demonstrated that the suspended cells began to aggregate to form chainlike clusters along the direction of the uniform AC electric field at an earlier stage of the field application. Moreover, the predicted results were similar to the experimental results. These findings indicate that the chain-like cell clustering arises from the long-range dipole-dipole interaction of neighboring cells, but under the action of the gravitational force that likely hinders the growth of clusters in the vertical direction.

Numerical study of oxygen transport in a carotid bifurcation.

This study investigates the oxygen mass transport in the region around the human carotid bifurcation, particularly addressing the effects of bifurcation geometry and pulsatile blood flow on the oxygen transport between the blood flow and artery wall tissue, coupled with the metabolic oxygen consumption and oxygen diffusion in the artery wall tissue. The temporal variations and spatial distributions of the oxygen tension are predicted quantitatively using a geometric model of the human carotid bifurcation and realistic blood flow waveforms. Results reveal that the flow separation at the outside wall of the sinus of the internal carotid artery (ICA) can markedly alter the flow pattern, oxygen tension and the oxygen wall flux. Results also clarify that the flow unsteadiness has a secondary effect on the oxygen tension inside the wall. The non-dimensional oxygen flux, the Sherwood number Sh, at the outside wall of the ICA sinus, takes markedly lower values of about 45 than at other sites because the rates of oxygen transport by the convective flow are reduced at the outside wall of the ICA sinus. The transverse distributions of the oxygen tension inside the artery wall show parabolic profiles having minima in the middle of the wall thickness, with the lowest value of 35 mmHg. These predicted distributions of the oxygen tension inside the wall closely resemble those obtained from experiments. The results demonstrate that hypoxic zones appear inside the artery walls at locations where atherosclerotic lesions are prone to develop.

Effect of the stress phase angle on the strain energy density of the endothelial plasma membrane.

Endothelial cells are simultaneously exposed to the mechanical forces of fluid wall shear stress (WSS) imposed by blood flow and solid circumferential stress (CS) induced by the blood vessel's elastic response to the pressure pulse. Experiments have demonstrated that these combined forces induce unique endothelial biomolecular responses that are not characteristic of either driving force alone and that the temporal phase angle between WSS and CS, referred to as the stress phase angle, modulates endothelial responses. In this article, we provide the first theoretical model to examine the combined forces of WSS and CS on a model of the endothelial cell plasma membrane. We focus on the strain energy density of the membrane that modulates the opening of ion channels that can mediate signal transduction. The model shows a significant influence of the stress phase angle on the strain energy density at the upstream and downstream ends of the cell where mechanotransduction is most likely to occur.

A novel single-photon counting technique applied to highly sensitive measurement of [Ca2+]i transient in human aortic smooth muscle cells.

This study demonstrates that aequorin, a luminescent natural dye, is useful for vascular cell intracellular Ca2+ concentration ([Ca2+]i) determination. A new single-photon counting technique was developed to resolve the effects of fluid flow shear stress on [Ca2+]i in human aortic smooth muscle cells (HASMCs). Confluent HASMCs were grown on petri dishes loaded with aequorin. Then the dishes were placed in a luminometer chamber after the physiological level of shear stress was applied to the HASMC surfaces. The chamber was housed inside a highly sensitive photomultiplier tube. It detected ultraweak photon emission in response to the [Ca2+]i transient. In the presence of 2.0 mM extracellular Ca2+, a shear stress of 12 dyn cm2, applied for 60 s to the top surface of the HASMC monolayer, elicited a sharp increase in [Ca2+]i.

Oxygen mass transport in a compliant carotid bifurcation model.

The purpose of the present study was to investigate oxygen mass transfer in the human carotid bifurcation, focusing on the effects of the wall compliance and flow field on the temporal variation and spatial distribution of the oxygen wall flux. Details of unsteady convective-diffusive oxygen transport were examined numerically using a compliant model of the human carotid bifurcation and realistic blood flow waveforms. Results reveal that axial flow separation at the outer common-internal carotid wall can significantly alter the flow field, oxygen tension field, and oxygen wall flux distribution. At the outer wall of the sinus, the Sherwood number, Sh (non-dimensional oxygen wall flux), takes on significantly lower values than at other sites due to the attenuation of transport rates by convective flow away from wall. More specifically, the lowest value of Sh was Sh approximately 6 (in the sinus), which is much lower than the value of the non-dimensional oxygen consumption rate (Damkohler number, Da) in the reactive wall tissue (Da=29-39). At the inner wall of the sinus, Sh approximately 170 is far above the expected value of Da. This implies that flow separation on the outer wall of the sinus provides a very strong fluid mechanical barrier to oxygen transport; whereas at the inner wall of the sinus, the mechanism of transport is controlled by the wall consumption rate.

Internal elastic lamina affects the distribution of macromolecules in the arterial wall: a computational study.

The internal elastic lamina (IEL), which separates the arterial intima from the media, affects macromolecular transport across the medial layer. In the present study, we have developed a two-dimensional numerical simulation model to resolve the influence of the IEL on convective-diffusive transport of macromolecules in the media. The model considers interstitial flow in the medial layer that has a complex entrance condition because of the presence of leaky fenestral pores in the IEL. The IEL was modeled as an impermeable barrier to both water and solute except for the fenestral pores that were assumed to be uniformly distributed over the IEL. The media were modeled as a heterogeneous medium composed of an array of smooth muscle cells (SMCs) embedded in a continuous porous medium representing the interstitial proteoglycan and collagen fiber matrix. Results for ATP and low-density lipoprotein (LDL) demonstrate a range of interesting features of molecular transport and uptake in the media that are determined by considering the balance among convection, diffusion, and SMC surface reaction. The ATP concentration distribution depends strongly on the IEL pore structure because ATP fluid-phase transport is dominated by diffusion emanating from the fenestral pores. On the other hand, LDL fluid-phase transport is only weakly dependent on the IEL pore structure because convection spreads LDL laterally over very short distances in the media. In addition, we observe that transport of LDL to SMC surfaces is likely to be limited by the fluid phase (surface concentration less than bulk concentration), whereas ATP transport is limited by reaction on the SMC surface (surface concentration equals bulk concentration).

Flow through internal elastic lamina affects shear stress on smooth muscle cells (3D simulations).

We describe a three-dimensional numerical simulation of interstitial flow through the medial layer of an artery accounting for the complex entrance condition associated with fenestral pores in the internal elastic lamina (IEL) to investigate the fluid mechanical environment around the smooth muscle cells (SMCs) right beneath the IEL. The IEL was modeled as an impermeable barrier to water flow except for the fenestral pores, which were assumed to be uniformly distributed over the IEL. The medial layer was modeled as a heterogeneous medium composed of a periodic array of cylindrical SMCs embedded in a continuous porous medium representing the interstitial proteoglycan and collagen matrix. Depending on the distance between the IEL bottom surface and the upstream end of the proximal layer of SMCs, the local shear stress on SMCs right beneath the fenestral pore could be more than 10 times higher than that on the cells far removed from the IEL under the conditions that the fenestral pore diameter and area fraction of pores were kept constant at 1.4 microm and 0.05, respectively. Thus these proximal SMCs may experience shear stress levels that are even higher than endothelial cells exposed to normal blood flow (order of 10 dyn/cm(2)). Furthermore, entrance flow through fenestral pores alters considerably the interstitial flow field in the medial layer over a spatial length scale of the order of the fenestral pore diameter. Thus the spatial gradient of shear stress on the most superficial SMC is noticeably higher than computed for endothelial cell surfaces.