A trachea-inspired bifurcated microfilter capturing viable circulating tumor cells via altered biophysical properties as measured by atomic force microscopy.

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 µL min(-1), an 8 µm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

Microslit on a chip: A simplified filter to capture circulating tumor cells enlarged with microbeads.

Microchips are widely used to separate circulating tumor cells (CTCs) from whole blood by virtues of sophisticated manipulation for microparticles. Here, we present a chip with an 8 µm high and 27.9 mm wide slit to capture cancer cells bound to 3 µm beads. Apart from a higher purity and recovery rate, the slit design allows for simplified fabrication, easy cell imaging, less clogging, lower chamber pressure and, therefore, higher throughput. The beads were conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) to selectively bind to breast cancer cells (MCF-7) used to spike the whole blood. The diameter of the cell-bead construct was in average 23.1 µm, making them separable from other cells in the blood. As a result, the cancer cells were separated from 5 mL of whole blood with a purity of 52.0% and a recovery rate of 91.1%, and also we confirmed that the device can be applicable to clinical samples of human breast cancer patients. The simple design with microslit, by eliminating any high-aspect ratio features, is expected to reduce possible defects on the chip and, therefore, more suitable for mass production without false separation outputs.

Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS.

We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.

A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads.

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.

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.

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter.

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 µm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.

Multistage-multiorifice flow fractionation (MS-MOFF): continuous size-based separation of microspheres using multiple series of contraction/expansion microchannels.

Previously we introduced a novel hydrodynamic method using a multi-orifice microchannel for size-based particle separation, which is called a multi-orifice flow fractionation (MOFF). The MOFF has several advantages such as continuous, non-intrusive, and minimal power consumption. However, it has a limitation that the recovery yield is relatively low. Although the recovery may be increased by adjusting parameters such as the Reynolds number and central collecting region, poor purity inevitably followed. We newly designed and fabricated a microfluidic channel for multi-stage multi-orifice flow fractionation (MS-MOFF), which is made by combining three multi-orifice segments, and consists of 3 inlets, 3 filters, 3 multi-orifice segments and 5 outlets. The structure and dimensions of the MS-MOFF were determined by the hydrodynamic principles to have constant Reynolds numbers at each multi-orifice segment. Polystyrene microspheres of two different sizes (7 µm and 15 µm) were tested. With this device, we made an attempt to improve recovery and minimize loss of purity by collecting and re-separating non-selected particles of the first separation. The final recovery successfully increased from 73.2% to 88.7% while the final purity slightly decreased from 91.4% to 89.1% (for 15 µm). These values were never achievable with the single-stage MOFF (SS-MOFF) having only one multi-orifice segment in our previous work. The MS-MOFF channel will be useful for clinical applications, such as separation of circulating tumor cells (CTC) or rare cells from human blood samples.