A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells

Abstract

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 μm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

“…[10][11][12] Cheung et al 9 investigated the characteristics of the detachment of the isolated cells under flow acceleration in a bio-functionalized micro-channel; in this study, detachment efficiency of $90% of bounded cells required a high shear stress $200 dyn/cm 2 , which significantly decreased cell viability and caused changes in gene expression. 3,18 Using bubble-induced detachment of affinity-adsorbed cells has been reported in recent years. 14,15,19 Surface attached particles may be released by introducing an air-water interface, which creates a net surface tension upon particle.…”

“…[1][2][3][4][5][6][7] Despite allowing for the rapid isolation of these clinically relevant cell types, microfluidic methods are facing the challenge of releasing and recovering the isolated target cells for further biological analysis. 8 Due to the nature of low concentration of CTCs in whole cellular population after separation process, the acceptable efficiency of releasing cells should approach 100% for broader applications such as sensitively monitoring therapy response, diagnosis of early stage cancer, and analysis of genetic makeup.…”

Emerging techniques in molecular detection of circulating tumor cells

“…A combined micromagnetic-microfluidic device has been developed to isolate, detect and culture viable CTCs from whole blood. CTCs can be easily removed from the device and expand in culture for additional analytical studies or potential drug sensitivity testing [24].…”

Microdevice in Cellular Pathology: Microfluidic Platforms for Fluorescence in situ Hybridization and Analysis of Circulating Tumor Cells

“…5 To enhance efficiency of detection, a magnetic beads system was integrated into the microchannel. [29][30][31] Karabacak et al 31 developed an isolation method of rare CTCs from blood samples by using deterministic lateral displacement (DLD) and magnetic beads with a leukocyte-specific antibody. The device was composed of two separate microfluidic devices: Module 1, using DLD to remove red cells, plasma, and free magnetic beads from the whole blood by size-based deflection; and Module 2, involving inertial focusing and magnetophoresis for the separation of bead-labeled WBCs and unlabeled CTCs (Fig.…”