Electrorotation measurements were used to demonstrate that the dielectric properties of the metastatic human breast cancer cell line MDA231 were significantly different from those of erythrocytes and T lymphocytes. These dielectric differences were exploited to separate the cancer cells from normal blood cells by appropriately balancing the hydrodynamic and dielectrophoretic forces acting on the cells within a dielectric affinity column containing a microelectrode array. The operational criteria for successful particle separation in such a column are analyzed and our findings indicate that the dielectric affinity technique may prove useful in a wide variety of cell separation and characterization applications.Cell separation has numerous applications in medicine, biotechnology, and research and in environmental settings. For example, the use of autologous bone marrow transplants in the remediation of advanced cancers requires the removal of cancer cells from the patient's marrow (1), the study of signaling between blood cells requires purified cell subpopulations (2), and the purification of contaminated water supplies necessitates elimination of parasites such as Giardia and Cryptosporidium (3,4). Current sorting technologies usually exploit differences in cell density, immunologic targets, or receptor-ligand interactions. These techniques are often inadequate, producing insufficiently pure cell populations, being too slow, or being too limited in the spectrum of target cells. Identification of novel properties by which different cell types may be discerned and of new ways for their selective manipulation are clearly fundamental components for improving sorting methodologies.A particle suspended in a medium of different dielectric characteristics becomes electrically polarized when subjected to an alternating electrical field. Interaction between this induced polarization and the field gives rise to various electrokinetic effects. For example, a spatially inhomogeneous field will exert a lateral dielectrophoretic (DEP) force on the particle, directing it toward the minimum of dielectric potential (5-9), while a rotating field will induce particle electrorotation (ROT; refs. 10-12). The frequency dependencies of the magnitude and direction of these forces are functions of the intrinsic electrical properties of the particle that depend on the particle constitution and structural organization (13-15). For living cells, these characteristics are defined by composition, morphology, and phenotype (16, 17). Thus, DEP and ROT have been used to study bacteria (18), yeasts (11,19), plant cells (10), and mammalian cells (20,21) and to investigate cellular alterations accompanying physiological changes such as mitotic stimulation (16) and induced differentiation (7,17,21 MATERIALS AND METHODS Cells. Peripheral blood was collected by venipuncture into 90 parts Ca2+/Mg2+-free phosphate-buffered saline containing 5 mM hemisodium EDTA to prevent clotting. T lymphocytes were obtained from (human immunodeficiency virusa...

The application of dielectrophoretic field-flow fractionation (depFFF) to the isolation of circulating tumor cells (CTCs) from clinical blood specimens was studied using simulated cell mixtures of three different cultured tumor cell types with peripheral blood. The depFFF method can not only exploit intrinsic tumor cell properties so that labeling is unnecessary but can also deliver unmodified, viable tumor cells for culture and/or all types of molecular analysis. We investigated tumor cell recovery efficiency as a function of cell loading for a 25 mm wide × 300 mm long depFFF chamber. More than 90% of tumor cells were recovered for small samples but a larger chamber will be required if similarly high recovery efficiencies are to be realized for 10 mL blood specimens used for CTC analysis in clinics. We show that the factor limiting isolation efficiency is cell-cell dielectric interactions and that isolation protocols should be completed within ~15 minutes in order to avoid changes in cell dielectric properties associated with ion leakage.

Recent measurements have demonstrated that the dielectric properties of cells depend on their type and physiological status. For example, MDA-231 human breast cancer cells were found to have a mean plasma membrane specific capacitance of 26 mF/m(2), more than double the value (11 mF/m(2)) observed for resting T-lymphocytes. When an inhomogeneous ac electric field is applied to a particle, a dielectrophoretic (DEP) force arises that depends on the particle dielectric properties. Therefore, cells having different dielectric characteristics will experience differential DEP forces when subjected to such a field. In this article, we demonstrate the use of differential DEP forces for the separation of several different cancerous cell types from blood in a dielectric affinity column. These separations were accomplished using thin, flat chambers having microelectrode arrays on the bottom wall. DEP forces generated by the application of ac fields to the electrodes were used to influence the rate of elution of cells from the chamber by hydrodynamic forces within a parabolic fluid flow profile. Electrorotation measurements were first made on the various cell types found within cell mixtures to be separated, and theoretical modeling was used to derive the cell dielectric parameters. Optimum separation conditions were then predicted from the frequency and suspension conductivity dependencies of cell DEP responses defined by these parameters. Cell separations were then undertaken for various ratios of cancerous to normal cells at different concentrations. Eluted cells were characterized in terms of separation efficiency, cell viability, and separation speed. For example, 100% efficiency was achieved for purging MDA-231 cells from blood at the tumor to normal cell ratio 1:1 x 10(5) or 1:3 x 10(5), cell viability was not compromised, and separation rates were at least 10(3) cells/s. Theoretical and experimental criteria for the design and operation of such separators are presented.

Dielectrophoretic field-flow-fractionation (DEP-FFF) was applied to several clinically relevant cell separation problems, including the purging of human breast cancer cells from normal T-lymphocytes and from CD34 + hematopoietic stem cells, the separation of the major leukocyte subpopulations, and the enrichment of leukocytes from blood. Cell separations were achieved in a thin chamber equipped with a microfabricated, interdigitated electrode array on its bottom wall that was energized with AC electric signals. Cells were levitated by the balance between DEP and sedimentation forces to different equilibrium heights and were transported at differing velocities and thereby separated when a velocity profile was established in the chamber. This bulk-separation technique adds cell intrinsic dielectric properties to the catalog of physical characteristics that can be applied to cell discrimination. The separation process and performance can be controlled through electronic means. Cell labeling is unnecessary, and separated cells may be cultured and further analyzed. It can be scaled up for routine laboratory cell separation or implemented on a miniaturized scale.Modern cell separation techniques 1,2 have been fundamental to many advances in cell biology, molecular genetics, biotechno-logical production, clinical diagnostics, and therapeutics. The most common of these techniques, centrifugation, 2 electrophoresis, 3 and both fluorescence-(FACS) 4 and magnetic-activated-cell sorting (MACS), 5-6 take advantage of differences in cell density, electrical charge, and immunological surface markers. Using these methods, investigators and clinicians are able to deplete particular cell populations (e.g., purge tumor cells from stem cell transplants) or enrich them (e.g., purify CD34 + stem cells from human blood).As these technologies have reached maturity, however, it has become more difficult to make fundamental improvements in separation resolution, cell purity, sample size, and device cost and portability. Therefore, novel physical methods by which different cell types may be discriminated and selectively manipulated are desirable. Besides offering additional avenues for cell identification, such methods should, ideally, allow us to enhance cell separation, exploit integrated microfluidic methods, separate microliter-size samples, reduce cost, and develop portable separation devices. To this end, cell dielectric properties have been explored through dielectrophoresis (DEP) and other AC electro-kinetic effects 7-15 for developing cell separation techniques. [16][17][18][19][20] Dielectrophoretic forces occur on cells when a nonuniform electrical field interacts with fieldinduced electrical polarization. 8,15 Depending on the dielectric properties of the cells relative to their suspending medium, these forces can be positive or negative, directing the cells toward strong or weak electrical field regions, respectively. 8,15,21 12,17,[22][23][24] differential DEP forces can be applied to drive their separation into puri...

We present the principle of cell characterization and separation by dielectrophoretic field-flow fractionation and show preliminary experimental results. The operational device takes the form of a thin chamber in which the bottom wall supports an array of microelectrodes. By applying appropriate AC voltage signals to these electrodes, dielectrophoretic forces are generated to levitate cells suspended in the chamber and to affect their equilibrium heights. A laminar flow profile is established in the chamber so that fluid flows faster with increasing distance from the chamber walls. A cell carried in the flow stream will attain an equilibrium height, and a corresponding velocity, based on the balance of dielectrophoretic, gravitational, and hydrodynamic lift forces it experiences. We describe a theoretical model for this system and show that the cell velocity is a function of the mean fluid velocity, the voltage and frequency of the signals applied to the electrodes, and, most significantly, the cell dielectric properties. The validity of the model is demonstrated with human leukemia (HL-60) cells subjected to a parallel electrode array, and application of the device to separating HL-60 cells from peripheral blood mononuclear cells is shown.

The separation and purification of human blood cell subpopulations is an essential step in many biomedical applications. New dielectrophoretic fractionation methods have great potential for cell discrimination and manipulation, both for microscale diagnostic applications and for much larger scale clinical problems. To discover whether human leukocyte subpopulations might be separable by such methods, the dielectric characteristics of the four main leukocyte subpopulations, namely, B- and T-lymphocytes, monocytes, and granulocytes, were measured by electrorotation over the frequency range 1 kHz to 120 MHz. The subpopulations were derived from human peripheral blood by magnetically activated cell sorting (MACS) and sheep erythrocyte rosetting methods, and the quality of cell fractions was checked by flow cytometry. Mean specific membrane capacitance values were calculated from the electrorotation data as 10.5 (+/- 3.1), 12.6 (+/- 3.5), 15.3 (+/- 4.3), and 11.0 (+/- 3.2) mF/m2 for T- and B-lymphocytes, monocytes, and granulocytes, respectively, according to a single-shell dielectric model. In agreement with earlier findings, these values correlated with the richness of the surface morphologies of the different cell types, as revealed by scanning electron microscopy (SEM). The data reveal that dielectrophoretic cell sorters should have the ability to discriminate between, and to separate, leukocyte subpopulations under appropriate conditions.

An important enabling factor for realising integrated micro fluidic analysis instruments for medical diagnostics purposes is front-end sample preparation. Dielectrophoresis is a method that offers great potential for cell discrimination and isolation for sample processing, and here we have applied it to the problem of isolating malaria-infected cells from blood. During development of the malarial pathogen, Plasmodium falciparum, increases occur in the ionic permeability of the plasma membrane of infected erythrocytes. When challenged by suspension in a low conductivity medium, infected cells lose internal ions while uninfected cells retain them. The resultant dielectric differences between infected and uninfected cells were exploited by dielectrophoretic manipulation in spatially inhomogeneous, travelling electrical fields produced by two types of microelectrode arrays. Parasitised cells of ring form or later stage from cultures and clinical specimens were isolated by steric dielectric field-flow-fractionation, focused at the centre of a spiral electrode array, and identified and counted. The dielectrophoretic methods require only a few micro litres of blood, and should be applicable to the production of small, low-cost automated devices for assessing parasite concentrations with potential applicability to drug sensitivity studies and the diagnosis of malaria. By simple adjustment of the electrical field parameters, other cell subpopulations that characterise disease, such as residual cancer cells in blood, can be similarly isolated and analysed.

Dielectrophoretic/gravitational field-flow fractionation (DEP/G-FFF) was used to separate cultured human breast cancer MDA-435 cells from normal blood cells mixed together in a sucrose/dextrose medium. An array of microfabricated, interdigitated electrodes of 50 microns widths and spacings, and lining the bottom surface of a thin chamber (0.42 mm H x 25 mm W x 300 mm L), was used to generate DEP forces that levitated the cells. A 10-microL cell mixture sample containing approximately 50,000 cells was introduced into the chamber, and cancerous and normal blood cells were levitated to different heights according to the balance of DEP and gravitational forces. The cells at different heights were transported at different velocities under the influence of a parabolic flow profile that was established in the chamber and were thereby separated. Separation performance depended on the frequency and voltage of the applied DEP field and the fluid-flow rate. It took as little as 5 min to achieve cell separation. An analysis of the DEP/G-FFF results revealed that the separation exploited the difference in dielectric and density properties between cell populations. The DEP/G-FFF technique is potentially applicable to many biological and biomedical problems, especially those related to microfluidic systems.