Biomedical Microdevices 2:1, 41±49, 1999 # 2000 Kluwer Academic Publishers, Boston. Manufactured in The Netherlands. Micro¯uidic Cell Separation by 2-dimensional Dielectrophoresis Giovanni De Gasperis,* Jun Yang, Frederick F. Becker, Peter R. C. Gascoyne,{ and Xiao-Bo Wang Department of Molecular Pathology, Box 89, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston TX 77030 USA Abstract. We describe a micro¯uidic device for separating cells according to their dielectric properties by combining 2-dimensional dielectrophoretic forces with ®eld-¯ow-fractionation. The device comprises a thin chamber in which a travelling-wave electrical ®eld is generated by a planar, multilayer microelectrode array at the bottom. Under the balance of gravitational and dielectrophoretic levitation forces, cells introduced into the device are positioned at different equilibrium heights in a velocity pro®le established inside the chamber, and thereby transported at different velocities by the ¯uid. Simultaneously, cells are subjected to a horizontal travelling- wave dielectrophoretic force that de¯ects them across the ¯ow stream. The 2-dimensional dielectrophoretic forces acting on cells and the associated velocities in the ¯uid-¯ow and travelling-®eld directions depend sensitively on cell dielectric properties. The responses of cultured MDA-435 human breast cancer, HL-60 human leukemia and DS19 murine erythroleukemia cells, and of peripheral blood mononuclear (PBMN) cells were studied as functions of the frequency and voltage of the applied electric signals, and of the ¯uid ¯ow rate. Signi®cant differences were observed between the responses of different cell types. Cell separation was demonstrated by the differential redistribution of MDA-435 and PBMN cells as they ¯owed through the device. The device can be readily integrated with other micro¯uidic compo- nents for microscale sample preparation and analysis. Key Words. cell sorting, travelling wave dielectrophoresis, ®led- ¯ow-fractionation, computer microvision Introduction There is increasing interest in developing integrated micro¯uidic systems capable of performing a sequence of chemical and biochemical reactions for analysis or synthesis (Cheng et al., 1998; Kricka, 1998; Marshall and Hodgson, 1998; van den Berg and Lammerink, 1998). These integrated microsystems made of elec- trical, mechanical and ¯uidic components have wide applications in areas such as biomedical research, clinical diagnosis, food pathogen detection and envir- onmental monitoring. For example, a micro¯uidic system can be used in the molecular analysis of cancer cells within a small volume of pathologic ¯uid (e.g., ascitic and pleural ¯uid) to provide cancer prognostic information. A critical necessity for micro- systems used to process cell samples is the ability to discriminate and sort cells according to characteristic phenotypes. Several cell separation techniques are used currently in biological laboratories, including isopycnic centrifuga- tion that exploits cell density properties, and ¯uorescent or magnetic activated cell sorting (Ormerod, 1994; Lea et al., 1990) that exploits cell immunological or receptor targets. Although powerful in the macroscopic world, these techniques do not readily scale to microsystem applications. A physical-force mechanism dependent on cell properties and effective on the microscopic scale is therefore desirable. Dielectrophoresis (DEP), which requires no mechanical contact with cells and is generated by microelectrodes, is an effective mechanism for manipulating cells in microstructures (e.g.,: Fuhr et al., 1995c; Schnelle et al., 1993; Pethig, 1996; Wang et al., 1997a; Washizu et al., 1990). It has been used for cell trapping (Fuhr et al., 1995a; Wang et al., 1993), levitation (Kaler and Jones, 1990), linear transport (Fuhr et al., 1995b; Huang et al., 1993; Masuda et al., 1987), and concentration (Markx et al., 1994; Wang et al., 1997b). Acting alone or in conjunction with other forces, DEP forces can be applied to separate cells of different properties, including enrichment of CD34  cells from bone marrow (Stephens et al., 1996), purging of cancer cells from blood (Becker et al., 1995; Gascoyne et al., 1997), separation of bacteria from blood (Cheng et al., 1998; Wang et al., 1993), separation of different microorganisms (Markx et al., 1994), and separation of viable cells from non-viable cells (Markx and Pethig, 1995). Recently, the technique of dielectrophoretic ®eld- ¯ow-fractionation has been demonstrated to exhibit high discriminatory abilities for cell separation (Huang et al., 1997; Markx et al., 1997; Yang et al., 1999). Here, we report a new approach to cell separation that combines 2-dimensional DEP effects generated by a non- uniform travelling wave electrical ®eld with ®eld-¯ow- *Now at Parco Scienti®co e Tecnologico d'Abruzzo, Centro Integrazione Microsistemi, Via Antica Arischia 1, 67100 L'Aquila, Italy. {Corresponding author. E-mail: peter@solace.mdacc.tmc.edu 41