CONTINUOUS CELL SEPARATION BY CHIP-BASED TRAVELING-WAVE DIELECTROPHORESIS AND LAMINAR FLOW Lei Wang1, 2, Min Guo1, Cheng-Jun Huang1, 3 and Jing Cheng1, 2 1 2

National Engineering Research Center for Beijing Biochip Technology, Beijing. China

Department of Biological Sciences & Biotechnology, Tsinghua University, Beijing,China 3

Department of Electronic Sciences & Technology, Huazhong University of Science & Technology, Wuhan, China

(Email: [email protected]) Abstract A novel method for continuous cell separation is developed by integrating traveling-wave dielectrophoresis (twDEP) and laminar flow on a single biochip-based device. The separation of heterogeneous population of different cells is based on the difference between their dielectrophoretic characteristics and densities. Our preliminary experiment proved this theory by demonstrating the separation of red blood cells from human Jurkat cells with this device. It can be envisaged that this method is potentially applicable to address many biological and biomedical problems, especially those related to sample preparation. Keywords: Cell separation, Dielectrophoresis, Laminar flow 1. Introduction It still remains a challenge to discriminate and separate cells fast and effectively in micro-fluidic systems. Several methods have been developed for on-chip cell separation in recent years based on dielectrophoresis, such as dielectrophoretic (DEP) migration/retention[1,2], dielectrophoretic field-flow-fractionation(DEP-FFF)[3], and DEP-based cytometry[4]. Here we report a novel device for continuous high discriminatory cell separation and demonstrate its applicability in cell sorting. 2. Device design The device was constructed by pressing the poly(dimethylsiloxane) (PDMS) cover

piece with pre-molded microstructures tightly against the glass substrate whose upper surface was plated with an array of parallel gold electrodes. The microstructures fabricated on the PDMS plate include a rectangular flow chamber connected with 3 micro-channeled inlets and 5 outlets. Driven by the syringe pump, the cell suspension was allowed to flow in from the central inlet and meanwhile the separation buffer flowed in from the two adjacent inlets. The flow rates for all three inports were the same. This arrangement allows the generation of a steady laminar-flow-field inside the flow chamber (Fig. 1). The orientation of the planar electrodes on the glass chip is in parallel with the flow direction of the fluid. TV MONITOR

CCD

SEPARATED CELLS PDMS FLOW CHAMBERR BUFFER SAMPLE twDEP MICROCHIP SIGNAL SOURCE

Fig. 1. Schematic of the cell separation device that combines the functions of both twDEP and laminar flow. When the travelling-wave was not applied, the cells were carried by the fluid and exit from the central outlet. By choosing cell suspending solution with appropriate conductivity and applying an appropriate phase-shifted AC signal to the electrode array, negative dielectrophoretic forces were generated to levitate cells suspended in the chamber, and the cells deflected by the twDEP forces perpendicular to the flow direction. Under the transversal twDEP forces, the cells were subjected to sustained deflection until

they exit the chamber. In principle, our device should allow the separation of any type of cells with subtle differences in their dielectric properties even though they may not be separated by conventional dielectrophoretic cell separating methods or other methods. 3. Results and discussion In the preliminary study, a mixture of cultured human Jurkat cells (human T-cell leukemia) and human red blood cells (RBCs) was chosen to characterize the method and prove its effectiveness in differentiating mixed cells. The experiment demonstrated that both types of cells flowed out in the same direction when the twDEP force was applied. However, the transversal migration rate of Jurkat cells was greater than that of RBCs. When the cells moved towards the outlets along with the fluid flow, they moved away from the original trajectory, evolved into two strands, and exited the chamber from two different outlets (Fig. 2). Compared with most of dielectrophoresis-based cell separation apparatus developed in recent years, the separation method presented here facilitated the continuous separation other than batch-mode separation reported by other researchers [1, 2]. Furthermore, the twDEP-based separation approach is such that the cell modification by stains or antibodies is unnecessary compared with traditional cell separating method, such as FACS and MACS. As far as the chip format is concerned, the device introduced here can be readily integrated with the subsequent components such as micro-DNA extracting chips and micro reactor chips to form a sample-to-answer system. Flow Direction

Fig. 2. (a) Mixture of cells carried by the fluid moving towards the outlets without the application of the twDEP force.

Flow Direction Human RBCs

Fig. 2. (b) Cells experiencing laminar

flow

forces

and

down-ward twDEP forces are on their midway to different outlets. Jurkat cells Jurkat Cells

were biased further away from the central line of the chamber under the influence of the combined forces. Fig.2. (c) The moment Jurkat

Human RBCs

cells and human RBCs were exiting the chamber from two different outlets.

Jurkat Cells

Outlets

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