Abtrennung von zirkulierenden Tumorzellen bei hohem Durchsatz durch dielektrophoretische Filtration
Analytische Chemie
Zusammenfassung der Projektergebnisse
Dielectrophoresis (DEP) is an electrokinetic particle manipulation technique that allows separation of biological cells according to specific properties. This project investigated the possibility to separate circulating tumor cells (CTC) from white blood cells (peripheral blood mononuclear cells, PBMC) via dielectrophoretic filtration at high throughput. The separation of CTC from PBMC is a challenging separation task and finding solutions helps in early detection of cancers and in predicting and adjusting treatment options. Due to differences in cell membrane capacitance, CTC show a different DEP response compared to PBMC. Aim of this project is to use dielectrophoretic forces to selectively filter CTC from cell mixtures in porous media. Microscopic pillar fields in microchannels were used as model porous media to observe the response of fluorescently labeled cells. Such a design allows easy observation under a microscope and thus to probe cell response to the electric field. It was clearly possible to show different DEP responses of K562 leukemia cells compared to PBMC. For instance, at 20 µS/cm conductivity and 5 kHz frequency of the applied AC field, K562 cells would be trapped (i.e., filtered) by DEP forces in the pillar field, whereas PBMC would only experience weak DEP forces and would thus not be trapped and washed out of the channel by a superimposed feed flow. This allows separation between both particle types. In order to achieve actual cell separation at high throughput, the same principle was applied in macroscopic mullite foams sandwiched between two electrodes. In this setup particle concentration was measured by fluorescence spectroscopy. A separation between both particle types, however, was not possible to observe in this project. The main reason for this was the loss of fluorescence expression during the separation process. This was caused by formation of electropores by the electric field which might also be accompanied by a loss of cell viability. The dyes used in this project simply diffused out of the cells during the separation. This phenomenon made a quantification of cell separation in both microchannels and macroscopic filters not possible. This was surprising as previous research works in the literature treated similar cells in DEP setups using comparable field and fluid parameters without suffering from either a severely decreased viability or loss of fluorescence expression. Future steps should be made in researching (by experiments, simulation and literature work) the reversible and irreversible electroporation of cells as a function of medium conductivity, osmolarity, salt content, applied voltage and cell type to be able to propose separator designs that cause a separation without causing a loss of viability or fluorescence expression. Further, the interplay between loss of fluorescence expression and loss of viability should be studied further and, in case cells stay viable after application of field, different fluorescent stains should be considered (for example, the CellBrite Fix Membrane Stain).
