Antibody-free isolation of circulating tumor cells by dielectrophoretic field-flow fractionation
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This work focuses on the integration of microfluidics and dielectrophoresis(DEP) with the principles of field flow fractionation (FFF) to create a continuous-flow isolator for rare and viable circulating tumor cells (CTCs) from peripheral blood mononuclear cells (PBMNs) drawn from cancer patients. The method exploits differences in the plasma membrane capacitances of tumor and blood cells, which correspond to differences in the membrane surface areas of these cell types. DEP-FFF was first adapted to measure cell membrane capacitance, cell density and deformability profiles of cell populations. These properties of the NCI-60 panel of cancer cell types, which represents the wide functional diversity of cancers from 9 organs and leukemia, were compared with the normal cell subpopulations of peripheral blood. In every case, the NCI-60 cells exhibited membrane capacitance characteristics that were distinct from blood and, as a result, they could be isolated from blood by DEP. The heightened cancer cell membrane capacitances correlated strongly with membrane-rich morphological characteristics at their growth sites, including cell flattening, dendritic projections, and surface wrinkling. Following harvest from culture and maintenance in suspension, cancer cells were found to shed cytoplasm and membrane area over time and the suspended cell populations developed considerable morphological diversity. The shedding changed the cancer cell DEP properties but they could still be isolated from blood cells. A similar shedding process in the peripheral blood could account for the surprisingly wide morphological diversity seen among circulating cells isolated from clinical specimens. A continuous flow DEP-FFF method was devised to exploit these findings by allowing CTCs to be isolated from the nucleated cells of 10 mL clinical blood specimens in 40 minutes, an extremely high throughput rate for a microfluidic-based method. Cultured cancer cells could be isolated at 70-80% efficiency using this approach and the isolation of CTCs from clinical specimens was demonstrated. The results showed that the continuous DEP-FFF method delivers unmodified, viable CTCs for analysis, is perhaps universally applicable to isolation of CTCs from different cancer types and is independent of surface antigens - making it suitable for cells lacking the epithelial markers used in currently accepted CTC isolation methods.