Cellular Separation via Dielectrophoretic Field-Flow-Fractionation
Keith Dvorkin
ABSTRACT
I present a brief review of a cellular separation technique known as dielectrophoretic field-flow-fractionation. The technique relies upon suspending different cells at different heights within a laminar flow profile. The differentially suspended cells, thus, travel at different speeds and elute from the flow chamber at different times resulting in successful separation. An alternate fabrication procedure than that commonly described in the literature is then proposed utilizing a micromolded polydimethylsiloxane elastomer cast over a thick photoresist. The proposed procedure decreases the number of steps in the fabrication meanwhile providing more precise control over the dimensions of the resultant device.
INTRODUCTION
The isolation and separation of distinct cell populations from a suspended mixture, a process known as fractionation, presents a challenging and routinely encountered problem in cell biology, molecular genetics, clinical diagnostics and therapeutics.1 The most commonly used technique for separation of cell populations is centrifugation, which relies on differences in cell density to cause layered sedimentation of different cell populations. This technique has served rather well for procedures designed to deplete or enrich certain cell populations in many settings, but as this and other successful fractionation techniques have reached maturity, progress in the separation resolution, cell purity, sample size, device cost, and portability have been relatively hard to come by.1 Dielectrophoretic field-flow-fractionation or (DEP-FFF) is a recently developed (1997)2 cell separation technique which relies on the intrinsic differences in cellular dielectric constants to suspend cells at different heights in a fluid experiencing laminar flow. Two cell populations suspended in a thin chamber, with their inherent difference in dielectric constant and corresponding difference in equilibrium height, would therefore, experience different velocities in the familiar parabolic velocity profile of laminar flow, and would thus exhibit different elution times from this chamber. The basic principle of DEP-FFF is illustrated in fig. 1. This technique is rather simple to implement, may be done so under sterile conditions, and also provides excellent separation resolution. Since it relies on intrinsic differences in cellular populations rather than externally generated cues such as in fluorescence- or magnetic-activated cell sorting, it offers additional comfort to the user by ensuring that the cells exposed in the DEP-FFF chamber are in their normal metabolic state after passing through the separation system.3 The DEP-FFF system thus has potential applications in the separation of cancer cells from bone marrow or mobilized blood in the treatment of advanced cancers in which autologous hematopoietic cell transplantation is required.1 The DEP-FFF system also finds use in detecting cancer cells circulating in the peripheral blood, a diagnostic tool in the early detection of cancer. Finally, DEP-FFF provides a cell separation technique that is accessible from the microscale, and one that me be serially implemented in integrated microfluidic analysis devices such as the so called micro-total-analysis-systems or (μ-TAS).1
Fig. 1.1 Schematic depiction of the operational principle of DEP-FFF. Particles (or cells) with distinctive dielectric constants will suspend themselves at different equilibrium heights within the parabolic velocity profile of the advancing liquid, and will thus travel at different velocities. This differential in the velocity of traveling cells results in different elution times for different cell populations resulting in separation of the populations.
THEORETICAL BACKGROUND (appropriately watered down)
Following Gayscoyne et al., fig. 1 shows the setup of a typical DEP-FFF chamber, and the forces acting on particles in solution. FDEPz is the dielectrophoretic levitation force provided from the interdigitated array of electrodes on the bottom surface of the chamber. Under AC electrical excitation, particles with a different dielectric coefficient than their suspending medium will experience a force due to the polarization of the particle. That force may be either positive (attractive) or negative (repulsive) and depends on a number of variables including the frequency of the excitation and the dielectric constants of the medium and particle. Additionally, this DEP force decreases with the height of the particle over the electrode array. Thus, when the frequency is adjusted such that the DEP force on a particular particle or set of particles is negative (repulsive) that particle will suspend itself over the electrode array at the exact height where the repulsive DEP force just balances the sedimentation force, Fsed, in the opposite direction. Since different cell populations exhibit different characteristic dielectric constants, the equilibrium height, heq, at which the sedimentation and DEP forces balance will be unique to a particular cell type. When subjected to a parabolic flow velocity profile, this height variation may be exploited to physically separate the cell populations. Equations exist which allow one to predict the equilibrium height of suspended particles, and one may therefore, predict the elution times for a specified chamber geometry and fluid flow rate however, for our purposes a general understanding of the underlying physics will suffice.
Different cells exhibit characteristic differences in dielectric constant due mainly to differences manifest in the morphology of the cell membrane. The membrane capacitance, directly related to the polarizability of the cell as a whole, is found to increase with area of the cell membrane.4 As different cells exhibit characteristic membrane textures, ie. some cells have smooth surfaces while others exhibit a characteristic degree of folding and ruffles, the membrane capacitance and polarizability vary accordingly. In addition differences in membrane composition inherent among different cell types in also thought to influence the cell dielectric constant further discriminating among cell types.4
EXAMPLE: Separation of normal T-lymphocytes from human breast cancer cells at a demanding ratio
To further demonstrate the principle of operation as well as to provide some operational data and detail from a working DEP-FFF system we will look at the performance of the device in a specific cellular separation problem, that of separating breast cancer cells from T-cells. As previously mentioned, this separation is clinically relevant as a screening tool in the early detection of cancer cells present in the peripheral bloodstream.
Fig. 21 Cell count vs. elution time for A) T-lymphocytes and B) MDA-436 human breast cancer cells as a function of applied frequency. Both cell populations demonstrated a narrow elution peak at 5kHz with similar elution times. However, as the frequency was increased above 10kHz the MDA-436 elution peak rapidly broadened while that of the T-lymphocytes remained relatively narrow.
Cell fractograms (cell counts as determined by flow cytometry) as a function of applied frequency and elution time are shown in figure 2 for both T-cells and breast cancer cells (MDA-436). One immediately notices that both cell populations exhibit a sharp peak at 5kHz centered about 5 min of elution time. However, as the applied frequency increases over 10kHz the MDA-436 peak rapidly broadens while the T-cell counts remain relatively narrow with respect to elution time. This broadening results mainly from the near zero or positive DEP forces experienced by the MDA-436 cells at frequencies above ~20kHz. These small forces levitate the cells at very small equilibrium heights and thus low velocities, or tend to trap the breast cancer cells at the electrodes, while allowing the T-lymphocytes which experience negative DEP forces to be eluted from the chamber. After the complete elution of the T-cells, the remaining breast cancer cells may then be released from the chamber by decreasing the applied frequency. Cells mixed at an initial ratio of 2:3 were separated within 11 minutes with purity above 92% at 30kHz.1 Additional cell mixtures have also been successfully separated using DEP-FFF with even higher purity.1-3, 5
FABRICATION PROCEDURE
Although the device depicted in fig. 1 is by no means complicated its fabrication nevertheless involves several time consuming steps that one may eliminate with a few simple modifications to the procedure. The DEP-FFF system shown in figure 1 consists of a gold microelectrode array on a glass substrate fabricated by standard photolithographic patterning and liftoff techniques. The electrodes have 50 μm spacing and width and are pattered onto a 50X50 mm glass substrate. A Teflon spacer 420 μm thick, cut in the center to provide the separation channel (25X388 mm), is then sandwiched between the electrode array and an additional top glass plate. The two glass plates have between them three 1.6 mm holes drilled such that fluid and cells may be infused and extracted through attached tubing, and the whole construction must be tightly clamped together to avoid fluid leakage.
My proposed alterations to the fabrication procedure would change this three-layer structure to a two-layer structure in which no holes need to be drilled, and forceful clamping is unnecessary. In addition, one may define the geometry of the separation channel with lithographic precision. The alterations hinge on two key materials which exhibit unique properties, properties which have made then invaluable in much current microfluidic research. These materials are the ultra-thick negative SU-8 photoresists (Microchem, MA) and polydimethylsiloxane (PDMS). PDMS is a remarkable material available from Dow Corning as a two part kit: silicone oil plus a curing agent. When the curing agent is mixed with the silicone oil, it serves to crosslink the long molecular chains of the oil into an insoluble network, forming an elastomer in the process. The resulting rubbery material, similar in feel to a superballI, is optically transparent down to 300 nm, and when used in micromolding applications such as those in microcontact-printing (G. Whitesides), PDMS has been shown to accurately replicate features down to the nanometer scale. The inherent elasticity of PDMS allows the material to easily form conformal contacts that are watertight at most pressures encountered in microfluidic systems, and also facilitates release from micromolds without damage. SU-8 is an equally remarkable material which has found extensive use in the field of MEMS and microfluidics for its ability to easily form high aspect ratio microstructures by doing nothing more than conventional contact photolithography. SU-8, available in different thickness formulations, is merely an extremely high viscosity negative photoresist. It may be spin coated onto substrates in thicknesses exceeding 200 μm in a single spin coating step. When exposed to i-line (365nm) UV light the resist rapidly crosslinks to form a chemically resistant and thermally stable epoxy, that develops in solution to form structures with nearly vertical sidewalls.5
Fig. 3. Schematic depiction of the proposed fabrication process for a simple DEP-FFF system. a) Bare Si wafer. b) Thick photoresist patterned atop Si. c) PDMS molded and cured over SU-8 on Si mold. Right: Section of the final device including syringe ports for the introduction and extraction on cell suspensions.
As one may envision from the unique properties of these two materials my proposed fabrication procedure involves casting PDMS prepolymer mixed with curing agent over a microfabricated mold consisting of SU-8 photoresist on silicon. After curing, the PDMS elastomer may then be peeled off of the mold and placed on top of an interdigitated electrode array on glass similar to that shown in figure 1. Since the PDMS forms watertight conformal contacts only gentle pressure will be required to keep the PDMS in place. In addition, there will be no need to drill infusion and extraction ports as PDMS is readily penetrated by syringes and forms self-sealing punctures. SU-8 micromolding is ideal for forming intricate fluidic patterns the kind which are likely necessary in microsystems such as the μ-TAS. The proposed fabrication procedure and a section of the final device is shown schematically in figure 3.
CONCLUSION
DEP-FFF is a versatile technique for cellular separation. By separating cells based on their intrinsic differences it ensures that the separated cells are not affected by the fractionation procedure, and are thus available after separation for further study. In addition the separation technique avoids the use of bulky hardware such as centrifuges and flow cytometers which have been found difficult to implement in the microscale. DEP-FFF is, therefore, an ideal technique for use in microfluidic applications such as the μ-TAS in which cell separation is a critical step in device processing.
REFERENCES
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