Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet ...microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl
, which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.
Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis ...and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 µL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.
The use of precision medicine for chemotherapy requires the individualization of the therapeutic regimen for each patient. This approach improves treatment efficacy and reduces the probability of ...administering ineffective drugs. To ensure accurate decision-making in a timely manner, anticancer drug efficacy tests must be performed within a short timeframe using a small number of cancer cells. These requirements can be satisfied via microfluidics-based drug screening platforms, which are composed of complex fluidic channels and closed systems. Owing to their complexity, skilled manipulation is required. In this study, we developed a microfluidic platform, to accurately perform multiple drug efficacy tests using a small number of cells, which can be conducted via simple manipulation. As it is a small, open-chamber system, a minimal number of cells could be loaded through simple pipetting. Furthermore, the extracellular matrix gel inside the chamber provides an in vivo-like environment that enables the localized delivery of the drugs to spontaneously diffuse from the channels underneath the chamber without a pump, thereby efficiently and robustly testing the efficacy and resistance of multiple drugs. We demonstrated that this platform enabled the rapid and facile testing of multiple drugs using a small number of cells (~ 10,000) over a short period of time (~ 2 days). These results provide the possibility of using this powerful platform for selecting therapeutic medication, developing new drugs, and delivering personalized medicine to patients.
In this study, a planar split-ring resonator (SRR)-based RF biosensor was developed for label-free detection of biomolecules such as the prostate cancer marker, prostate specific antigen (PSA), and ...cortisol stress hormone. The biosensor has a resonance-assisted transducer and is excited by a time-varying magnetic field component of a local high-impedance microstrip line. The resulting device exhibits an intrinsic S21 resonance with a quality-factor (or Q-factor) of 50. For the biomolecular interaction, anti-PSA and anti-cortisol were immobilized on the gold surface of the resonator by a protein-G mediated bioconjugation process and corresponding frequency shifts of Δf1p=30±2 MHz (for anti-PSA) and Δf1c=20±3 MHz (for anti-cortisol) were observed. The additional frequency shift of each PSA and cortisol antigen with a 100pg/ml concentration was about 5±1.5MHz and 3±1MHz, respectively. From the experimental results, we confirmed that our device is very effective RF biosensor with a limit of detection (LOD) of 100pg/ml and has sufficiently feasibility as a label-free biosensing scheme.
Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. ...However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.
Abstract 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.
Selective cell enrichment technologies can play an important role in both diagnostic and therapeutic areas. However, currently used cell sorting techniques have difficulties in rapidly isolating only ...the desired target cells from a large volume of body fluids. In this work, we developed a filtering system that can quickly separate and highly concentrate cells from a large volume of solution, depending on their size, using a silicon membrane filter. To overcome the problems caused by material limitations of the brittle silicon, we designed a novel membrane filter with various pore designs. From these designs, the most optimal design with high pore density, while preventing crack formation was derived by applying fluid dynamics simulation and near-field stress analysis. The membrane filter system using the selected design was fabricated, and cell filtration performance was evaluated. The LNCaP cell in horse blood was recovered up to 86% and enriched to 187-fold compared to initial cell populations after filtration at a flow rate of 5 mL/min. The results demonstrate that the filter presented in this study can rapidly and selectively isolate target cells from a large volume of body fluid sample.
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.
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.
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