Fully Automated Circulating Tumor Cell Isolation Platform with Large-Volume Capacity Based on Lab-on-a-Disc

Abstract

Full automation with high purity for circulating tumor cell (CTC) isolation has been regarded as a key goal to make CTC analysis a "bench-to-bedside" technology. Here, we have developed a novel centrifugal microfluidic platform that can isolate the rare cells from a large volume of whole blood. To isolate CTCs from whole blood, we introduce a disc device having the biggest sample capacity as well as manipulating blood cells for the first time. The fully automated disc platform could handle 5 mL of blood by designing the blood chamber having a triangular obstacle structure (TOS) with lateral direction. To guarantee high purity that enables molecular analysis with the rare cells, CTCs were bound to the microbeads covered with anti-EpCAM to discriminate density between CTCs and blood cells and the CTCs being heavier than blood cells were only settled under a density gradient medium (DGM) layer. To understand the movement of CTCs under centrifugal force, we performed computational fluid dynamics simulation and found that their major trajectories were the boundary walls of the DGM chamber, thereby optimizing the chamber design. After whole blood was inserted into the blood chamber of the disc platform, size- and density-amplified cancer cells were isolated within 78 min, with minimal contamination as much as approximately 12 leukocytes per milliliter. As a model of molecular analysis toward personalized cancer treatment, we performed epidermal growth factor receptor (EGFR) mutation analysis with HCC827 lung cancer cells and the isolated cells were then successfully detected for the mutation by PCR clamping and direct sequencing.

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

“…We previously reported the development of CTC-isolation methods, based on changes in physical properties such as size and following CTC complexation with magnetic beads19202122. CTC-magnetic bead complexes could be isolated with high yield and purity by centrifugation through density-gradient media.…”

“…After vertical separation of CTC-magnetic bead complexes by magnetic force, CTC-magnetic bead complexes could be moved to the collection well by placing the magnet under the collection well. After collecting CTC-magnetic bead complexes using a micropipette, they were injected into a microchip filter for enumeration1920212223.…”

“…1. The isolation technique was based on the high-density and magnetic properties of CTC-magnetic microbead complexes, which were reported previously19202122. CTC-magnetic microbead complexes could be separated vertically in modified well-plates via magnetic force, due to their high density and magnetic properties (Fig.…”

Development of the automated circulating tumor cell recovery system with microcavity array

“…Anti-EpCAM antibody-coated magnetic particles are used in immunomagnetic separation-based systems such as CellSearch system (Velidex, Raritan, NJ) that has been approved by FDA for the diagnosis of breast, prostate and colon cancers Xie et al, 2014;Xu et al, 2011). Many reports have been published on antibody-coated tools including microfluidic channel, needle, and magnetic nanoparticles with the aid of the antibodies, which recognize CTC specific markers (Durgadas et al, 2011;Kamande et al, 2013;Kirby et al, 2012;Nora Dickson et al, 2011;Park et al, 2014;Svensson et al, 2014;Wang et al, 2004). However, this assay format has a limitation to apply the basic protocol to various types of tumor cells because the expression level of surface makers, especially EpCAM (Punnoose et al, 2010), was varied with tumor types.…”

Microfluidic device for sheathless particle focusing and separation using a viscoelastic fluid

“…Additionally, the throughput of our device can be enhanced by using a viscoelastic fluid with low viscosity but high elasticity, such as DNA [19] or hyaluronic acid (HA) solutions [20]. Therefore, the device could potentially be further developed as a clinical tool for high-throughput separation of targeted rare cells such as circulating tumor cells [42] and/or bacteria [43] from a biological sample.…”