Hui-Sung Moon

Continual collection and re-separation of circulating tumor cells from blood using multi-stage multi-orifice flow fractionation

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer; as such, the ability to isolate and quantify CTCs is of great biomedical importance. This research presents a multi-stage multi-orifice flow fractionation (MS-MOFF) device formed by combining three single-stage multi-orifice segments designed for separating breast cancer cells from blood. The structure and dimensions of the MS-MOFF were determined by hydrodynamic principles to have consistent Reynolds numbers (Re) at each multi-orifice segment. From this device, we achieved improved separation efficiency by collecting and re-separating non-selected target cells in comparison with the single-stage multi-orifice flow fractionation (SS-MOFF). The recovery of breast cancer cells increased from 88.8% to greater than 98.9% through the multi-stage multi-orifice segments. This device can be utilized to isolate rare cells from human blood, such as CTCs, in a label-free manner solely through the use of hydrodynamic forces.

Continuous labeling of circulating tumor cells with microbeads using a vortex micromixer for highly selective isolation.

Circulating tumor cells (CTCs) are identified in transit within the blood stream of cancer patients and have been proven to be a main cause of metastatic disease. Current approaches for the size-based isolation of CTCs have encountered technical challenges as some of the CTCs have a size similar to that of leukocytes and therefore CTCs are often lost in the process. Here, we propose a novel strategy where most of the CTCs are coated by a large number of microbeads to amplify their size to enable complete discrimination from leukocytes. In addition, all of the microbead labeling processes are carried out in a continuous manner to prevent any loss of CTCs during the isolation process. Thus, a microfluidic mixer was employed to facilitate the efficient and selective labeling of CTCs from peripheral blood samples. By generating secondary vortex flows called Taylor-Gortler vortices perpendicular to the main flow direction in our microfluidic device, CTCs were continuously and successfully coated with anti-epithelial cell adhesion molecule-conjugated beads. After the continuous labeling, the enlarged CTCs were perfectly trapped in a micro-filter whereas all of the leukocytes escaped.

Highly dense, optically inactive silica microbeads for the isolation and identification of circulating tumor cells.

Efficient isolation of circulating tumor cells (CTCs) from whole blood is a major challenge for the clinical application of CTCs. Here, we report an efficient method to isolate CTCs from whole blood using highly dense and transparent silica microbeads. The surfaces of silica microbeads were fully covered with an antibody to capture CTCs, and blocked by zwitterionic moieties to prevent the non-specific adsorption of blood cells. Owing to the high density of the silica microbeads, the complexation of CTCs with silica microbeads resulted in the efficient sedimentation of CTC-microbead complexes, which enabled their discrimination from other blood cells in density gradient media. Model CTCs (MCF-7, HCC827, and SHP-77) with various levels of epithelial cell adhesion molecule (EpCAM) were isolated efficiently, especially those with low EpCAM expression (SHP-77). Moreover, the transparency of silica microbeads enabled CTCs to be clearly identified without interference caused by microbeads. The improved sensitivity resulted in increased CTC recovery from patient samples compared with the FDA-approved CellSearch system (14/15 using our method; 5/15 using the CellSearch system). These results indicate that the isolation method described in this report constitutes a powerful tool for the isolation of CTCs from whole blood, which has important applications in clinical practice.

Vertical Magnetic Separation of Circulating Tumor Cells for Somatic Genomic-Alteration Analysis in Lung Cancer Patients.

Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients’ blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.

Micro-slit filter for separation of circulating tumor cells with high recovery and high purity

We present a novel method for separating circulating tumor cells (CTCs) with high recovery and purity at the same time using a micro-slit filter chip and a fully automated fluidic system. Considering white blood cells (WBCs) as big as CTCs are also captured with CTCs during filtration, we amplified the size of CTCs specifically using microbeads (3 μm) coated with anti-Epithelial Cell Adhesion Molecule (anti-EpCAM) to increase the size difference between WBCs and CTCs. The average diameter of MCF-7 cells was increased from 16.5 μm to 23.1 μm. A micro filter chip having an extremely high aspect ratio (AR=3488) rectangular slit was designed to prevent clogging which induces unwanted aggregation, capturing of other small blood cells and consequently decreasing purity. A fully automated fluid control system was implemented for the better reproducibility and the minimization of handling errors. The procedures from blood loading to staining, prior to analysis, were performed automatically. With the optimized condition, separation experiments using 5ml of normal whole blood spiked with 100 MCF-7 cells have demonstrated the reduction of clogging, high recovery (91.1 %) and high purity (52.0 %) at the same time.