Cell Separation by Dielectrophoretic Field-flow-fractionation

Abstract

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...

Tuning direct current streaming dielectrophoresis of proteins

“…It is worthy to point out that DEP trapping, i.e., the accumulation of particles at a specific location, is not always required for analytical applications. Field-flow fractionation based on DEP [34][35][36][37] or sorting at restrictions 38 are examples, in which DEP trapping is not necessary. Indeed, slight differences in DEP behavior are sufficient in order to take advantage of biomolecule DEP for separation.…”

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

“…In our experience, variability in DEP-FFF-based cell fractionation is principally of a biological nature and not due to intrinsic system or hardware variation. 3,27,42 Biological variance is manifest in DEP-FFF elution profiles as a shift in elution time and/ or peak broadening; however, a standardized protocol for cell processing and DEP-FFF enrichment can attenuate these effects. In the stromal vascular fraction enrichment experiments described here, the erythrocyte peak elution time is used as a standard reference point when comparing different runs from the same patient sample.…”

“…Our current generation DEP-FFF device is ideally suited for batch-mode separation and recovery of moderate quantities of cells (<10 6 cells per run). [25][26][27] The DEP-FFF device consists of a thin flow channel with microelectrodes patterned along the bottom surface. When energized, the electrodes provide a height-dependent dielectrophoretic force that is perpendicular to the flow axis and applied continuously along the length of the channel-sufficient differences in the density and dielectric characteristics of various cell types cause them to be positioned in different flowvelocity lamina, thereby resulting in differential elution from the separation chamber.…”

Development of three-dimensional integrated microchannel-electrode system to understand the particles' movement with electrokinetics

“…16 In microfluidic systems, a pressure-driven flow by electroosmotic force is often applied to deliver the sample or cells into the target region. 17 Exploiting the resultant effect of the combination of the above electrokinetic forces was used as field-flow fractionation (FFF) 18 by Kuczenski et al, 19 Gielen et al, 20 Wang et al, 21 and Yasukawa et al 22 to manipulate the particles in two dimensional scope, while the electrokinetic forces generated from a simple two dimensional planner electrodes. The overall mechanism of these forces in 2D is straightforward.…”