Deploying an Orthogonal Turn for Isolation of Rare Cell Populations from Vascular Environments

Abstract

Cancer cells are shed from metastatic primary tumors, bearing the potential for blood-borne metastasis to distant vital organs. Metastasis is predominantly responsible for cancer-related deaths. These circulating tumor cells (also known as CTCs) are highly invasive and can be present in the vasculature as single cells or as clusters. CTCs have been proposed as an important biomarker to assess the aggressiveness of cancer, the effectiveness of the treatment, and disease progression. Although most of the cells have epithelial receptors on their surface, such as Epithelial Cell Adhesion Molecules (EpCAMs), the molecular diversity on the surfaces of such cells is still not completely characterized. The circulating tumor cells (or CTCs) tend to be larger in size and higher in density with respect to the rest of the blood cells and in their density. CTCs are extraordinarily rare, i.e., one among a billion blood cells which make their isolation difficult.

Existing technologies carry out CTC separation by either inducing external forces (active separation) or using intrinsic hydrodynamic forces (passive separation). In active separation, the external forces have to be larger than the flow-induced forces, which results in a limited throughput. Moreover, these techniques often involve biomarkers and labelling agents (usually EpCAM antibodies), which not only puts a question on the viability of the captured cells but also might fail to work for CTCs that do not express such markers. The passive separation method is carried out by simply controlling the hydrodynamic properties of the flow. Of these, inertial microfluidics separation has high throughput, whereby large sample volumes can be processed in a short time. High throughput vortex trapping and CTC separation has been described by Di Carlo et al. in their vortex-chip technology. However, the operation of their device requires drastically high flow velocities (particle velocity ̴ 4 m/s), which are prone to damage the cells and affect their viability.

In this dissertation, an inertial microfluidic vortex chip incorporating an orthogonal turn is investigated for the isolation and separation of CTCs. These chips function at significantly lower (38% of previously reported) flow velocities. Fluid flowing through the chip is constrained to exit the trapping chamber at right angles to that of its entry. Such a flow configuration leads to the formation of a vortex in the chamber and above a critical flow velocity, larger particles are trapped in the vortex, whereas smaller particles get ejected with the flow: we call this phenomenon the turn-effect.

I explain how different forces contribute to the turn-effect in the orthogonal design by acting on cells, and pushing them into specific vortices in a size- and velocity-dependent fashion. Furthermore, we have characterized the critical velocities for trapping particles of different sizes on chips with distinct entry-exit configurations. Optimal architectures for stable vortex trapping at low flow velocities are identified using polystyrene beads and blood cells.

Subsequently, I demonstrated selective trapping of human breast cancer cells mixed with whole blood at low concentrations. An isolation protocol to separate the trapped particles was developed and optimized on a scaled-up device that uses serialization and parallelization. After isolating spiked circulating cancer cells from diluted blood, we were also able to culture them.

In summary, a label-free inertial microfluidic vortex trapping setup incorporating an orthogonal turn was developed and optimized for the size-based gentle separation of CTCs which are larger and rarer than other blood cells. Some further design modifications were also suggested in the latter part of the work to increase the efficiency of enrichment.