(322f) High Throughput Microfluidic Separation of Tumor Initiating Cells (TICs) Using Contactless Dielectrophoresis

Tumor Initiating Cells (TICs), also known as Cancer Stem Cells, are a finite population of cancer cells possessing the unique ability to transplant a new tumor from an existing one and are putatively responsible for the metastatic properties of tumors. Contactless-dielectrophoresis (cDEP) is a non-invasive marker free technique able to characterize and enrich rare cells, such as TICs suspended in a low conductivity medium. cDEP separation is based on physical and electrical properties, and is independent of cell genotype. cDEP alleviates limitations of classical dielectrophoresis by using external electrodes placed in highly conductive channels which are separated from the sample by a thin insulating layer. We have developed the chip technology to make it more robust with increased throughput and increased yield for taking the enriched population off-chip.

When designing DEP separation devices the typical assumption is that the density of the cell suspension is low enough that cell-to-cell interactions can be neglected. That may be true for continuous flow through DEP devices however it is not true for DEP devices that trap cells on posts in areas with a high electric field. The force between induced dipoles (cells) contributes to the DEP force on a single cell level and pearl chaining is difficult to avoid. Therefore trapping depends not only on the trapping voltage of specific cells, but also on cell density (cells per ml). A possible solution that reduces this issue is to use a very low density of cells (~10⁵ cells/ml), however throughput of such an approach is low. A better solution is to make the trapping area small enough that only one cell gets trapped while other cells are carried away by a sufficiently fast flow of media. A cDEP device was designed for trapping only one or two cells on a post at a time while maintaining high throughput. The diameter of the insulating pillars to make the electric field non-uniform were optimized to 20 μm which is close to the cell diameter. Due to the hydrodynamic drag force typically only up to two cells can trap on one pillar therefore pearl chaining and non-specific trapping is greatly reduced. The resulting high throughput separation device has about 17,000 insulating pillars 80 μm apart where cells get trapped with a main channel that is 12.3 mm long, 7.6 mm wide and 50 μm high. The developed protocol for separation is to pump cells suspended in DEP buffer at 2.5 μl/min and trap them at 300 Vrms, 100 kHz. When the insulating pillars are saturated with cells, syringes at the inlet are switched and pure DEP buffer is pumped into the device to wash non-trapped cells down the channel to the outlet for collection. The voltage is then gradually lowered, and cells are selectively released and separated cells are collected in tubes at the output. Separated cells are counted and analyzed. For assessing spheroid formation ability, a device with 4 parallel channels was used, providing enough separated cells for culturing in low-attachment dishes or alternatively cells can be analyzed by flow cytometer for expression of markers such as ALDH.