The removal of human breast cancer cells from hematopoietic CD34+ stem cells by dielectrophoretic field-flow-fractionation

Abstract

Dielectrophoretic field-flow-fractionation (DEP-FFF) was used to purge human breast cancer MDA-435 cells from hematopoietic CD34+ stem cells. An array of interdigitated microelectrodes lining the bottom surface of a thin chamber was used to generate dielectrophoretic forces that levitated the cell mixture in a fluid flow profile. CD34+ stem cells were levitated higher, were carried faster by the fluid flow, and exited the separation chamber earlier than the cancer cells. Using on-line flow cytometry, efficient separation of the cell mixture was observed in less than 12 min, and CD34+ stem cell fractions with a purity >99.2% were obtained. The method of DEP-FFF is potentially applicable to many biomedical cell separation problems, including microfluidic-scale diagnosis and preparative-scale purification of cell subpopulations.

FIG. 1

DEP-FFF experimental setup. Microfabricated electrodes on the bottom wall of the separation chamber were energized with electrical signals and provided DEP levitation forces. After being introduced to the chamber through the injection valve, the cells of different types in a mixture were levitated to different equilibrium heights under the balance of DEP and sedimentation forces. A fluid-flow profile was produced in the chamber from the injection syringe pump. The cells were transported through the chamber at different velocities corresponding to their heights, exited the chamber from the bottom outlet port, and were detected by the flow cytometer.

FIG. 2

Frequency dependency of DEP-FFF elution fractograms for (A) MDA-435 human breast cancer cells and (B) CD34⁺ stem cells obtained by the flow cytometer. Cells were suspended at 1.5 × 10⁶/ml in the sucrose buffer having an electrical conductivity of 10 mS/m. The applied voltage was 4 V p-p. The injection and withdrawal syringe pumps were operated at 2 and 1.8 ml/min, respectively.

FIG. 3

DEP-FFF fractograms for separating MDA-435 cells from CD34⁺ cells using the trap-and-release protocol. DEP field was operated at 40 kHz for 7 min and switched to 5 kHz for 7 min. CD34⁺ cells were prelabeled with PE-conjugated CD34 antibodies and were identified by flow cytometer to elute the chamber earlier than MDA-435 cells. DEP signal voltage and fluid-flow conditions were the same as those used for Figure 2.

FIG. 4

(A) DEP-FFF fractogram for separating MDA-435 cells from CD34⁺ cells by the swept-frequency protocol. (B) Corresponding contour plot for fluorescence level vs. elution time for cells that exited the DEP-FFF chamber. The DEP field was swept between 15 and 35 kHz for 7 min and then switched to 5 kHz for 7 min. DEP signal voltage and fluid-flow conditions were the same as those used in Figure 2.

FIG. 5

(A) Typical electrorotation spectra for CD34⁺ (circles) and MDA-435 (squares) cells in the sucrose buffer having a conductivity of 56 mS/m. Continuous curves show best fit of the single-shell dielectric model (30,31). (B) The frequency spectra of α_DEP for CD34⁺ (solid) and MDA-435 (broken line) cells under separation conditions (conductivity 10 mS/m) calculated using the dielectric parameters (Table 1) derived from ROT measurements.