Swee Jin Tan

Microfluidics for cell separation.

The need for efficient cell separation, an essential preparatory step in many biological and medical assays, has led to the recent development of numerous microscale separation techniques. This review describes the current state-of-the-art in microfluidics-based cell separation techniques. Microfluidics-based sorting offers numerous advantages, including reducing sample volumes, faster sample processing, high sensitivity and spatial resolution, low device cost, and increased portability. The techniques presented are broadly classified as being active or passive depending on the operating principles. The various separation principles are explained in detail along with popular examples demonstrating their application toward cell separation. Common separation metrics, including separation markers, resolution, efficiency, and throughput, of these techniques are discussed. Developing efficient microscale separation methods that offering greater control over cell population distribution will be important in realizing true point-of-care (POC) lab-on-a-chip (LOC) systems.

Microdevice for the isolation and enumeration of cancer cells from blood

Cancer metastasis is the main attribute to cancer-related deaths. Furthermore, clinical reports have shown a strong correlation between the disease development and number of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. Here, we present a label-free microdevice capable of isolating cancer cells from whole blood via their distinctively different physical properties such as deformability and size. The isolation efficiency is at least 80% for tests performed on breast and colon cancer cells. Viable isolated cells are also obtained which may give further insights to the understanding of the metastatic process. Contrasting with conventional biochemical techniques, the uniqueness of this microdevice lies in the mechanistic and efficient means of isolating viable cancer cells in blood. The microdevice has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting.

Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients

The isolation of circulating tumor cells (CTCs) using microfluidics is attractive as the flow conditions can be accurately manipulated to achieve an efficient separation. CTCs are rare events within the peripheral blood of metastatic cancer patients which makes them hard to detect. The presence of CTCs is likely to indicate the severity of the disease and increasing evidences show its use for prognostic and treatment monitoring purposes. We demonstrated an effective separation using a microfluidic device to utilize the unique differences in size and deformability of cancer cells to blood cells. Using physical structures placed in the path of blood specimens in a microchannel, CTCs which are generally larger and stiffer are retained while most blood constituents are removed. The placements of the structures are optimized by computational analysis to enhance the isolation efficiency. With blood specimens from metastatic lung cancer patients, we confirmed the successful detection of CTCs. The operations for processing blood are straightforward and permit multiplexing of the microdevices to concurrently work with different samples. The microfluidic device is optically transparent which makes it simple to be integrated to existing laboratory microscopes and immunofluorescence staining can be done in situ to distinguish cancer cells from hematopoietic cells. This also minimizes the use of expensive staining reagents, given the small size of the microdevice. Identification of CTCs will aid in the detection of malignancy and disease stage as well as understanding the phenotypic and genotypic expressions of cancer cells.

Microfluidic enrichment for the single cell analysis of circulating tumor cells

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

Molecular Mechanistic Insights into the Endothelial Receptor Mediated Cytoadherence of Plasmodium falciparum-Infected Erythrocytes

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the ‘holder’ for providing stable binding.

Manipulation and isolation of single cells and nuclei.

The heterogeneous behavior of cells within a cell population makes measurements at the multicellular level insensitive to changes in single cells. Single-cell and single-nucleus analyses are therefore important to address this deficiency which will aid in the understanding of fundamental biology at both the cellular and subcellular levels. Recent technological advancements have enabled the development of new methodologies capable of handling these new challenges. This review highlights various techniques used in single-cell and single-nucleus manipulation and isolation. In particular, the applications related to microfluidics, electrical, optical, and physical methods will be discussed. Ultimately, it is hoped that these techniques will enable fundamental tests to be conducted on single cells and nuclei. One important potential outcome is that this will contribute not only towards detection and isolation of diseased cells but also more accurate diagnosis and prognosis of human diseases.

A Microfluidic Device to Sort Cells Based on Dynamic Response to a Stimulus

Single cell techniques permit the analysis of cellular properties that are obscured by studying the average behavior of cell populations. One way to determine how gene expression contributes to phenotypic differences among cells is to combine functional analysis with transcriptional profiling of single cells. Here we describe a microfluidic device for monitoring the responses of single cells to a ligand and then collecting cells of interest for transcriptional profiling or other assays. As a test, cells from the olfactory epithelium of zebrafish were screened by calcium imaging to identify sensory neurons that were responsive to the odorant L-lysine. Single cells were subsequently recovered for transcriptional profiling by qRT-PCR. Responsive cells all expressed TRPC2 but not OMP, consistent with known properties of amino-acid sensitive olfactory neurons. The device can be adapted for other areas in biology where there is a need to sort and analyze cells based on their signaling responses.

Microdevice for Trapping Circulating Tumor Cells for Cancer Diagnostics

Cancer metastasis is the main attribute to cancerrelated deaths and early detection is one of the most effective means to treat the disease. Spreading of cancer to distant sites is achieved usually through the circulatory system and the number of circulating tumor cells (CTCs) in peripheral blood is strongly associated to cancer development. Thus detecting CTCs in blood samples will be helpful in identifying susceptible subjects. Here, we propose a label-free technique to isolate these cancerous cells from blood. Using cancer cells spiked in blood samples, isolation efficiency of at least 80% were obtained with high isolation purity. The microdevice also preserved the integrity of the isolated cells to allow further studies to be done on these cells. These results showed a potential effective microdevice for CTCs studies and possible clinical application.

Personalized Treatment Through Detection and Monitoring of Genetic Aberrations in Single Circulating Tumor Cells

Circulating tumor cells (CTCs) present a viable alternative to access tumor materials other than primary biopsies in cancer. This disease is among the most widespread in the world and is difficult to target because of its complex nature, challenges in getting quality samples and dynamic temporal changes in response to treatment. Conventional methods of detection and monitoring the disease profile do not suffice to be able to target the heterogeneity that exists at the cellular level. CTCs have been identified as a possible substitute for tumor tissue samples, and can be used to complement current disease management. Challenges in CTCs molecular analysis lie in the purity of the sample, which is masked by the presence of large quantities of white blood cells (WBCs) . In this chapter, we present a microfluidic biochip platform that performs secondary purification to isolate single CTCs efficiently. Studying single CTCs will allow for sensitive detection of critical mutations and addressing intercellular variances that will be otherwise missed easily due to low mutation frequencies when evaluating bulk cell retrieval. Using the biochip, we isolated single CTCs, and conducted personalized integrated EGFR mutational analysis using conventional polymerase chain reaction (PCR) and Sanger sequencing. We also demonstrated that high quality next generation sequencing (NGS) libraries can be readily generated from these samples. In our initial study, we revealed that the dominant EGFR mutations such as L858R and T790M could be detected in Non Small Cell Lung Cancer (NSCLC) patients with low CTC counts. We envision the biochip will enable efficient isolation of rare single cells from samples. This technology coupled with downstream molecular characterization of CTCs will aid in realizing the personalized medicine for cancer patients.

An extracellular matrix-related prognostic and predictive indicator for early-stage non-small cell lung cancer

The prognosis and prediction of adjuvant chemotherapy (ACT) response in early-stage non-small cell lung cancer (NSCLC) patients remain poor in this era of personalized medicine. We hypothesize that extracellular matrix (ECM)-associated components could be potential markers for better diagnosis and prognosis due to their differential expression in 1,943 primary NSCLC tumors as compared to 303 normal lung tissues. Here we develop a 29-gene ECM-related prognostic and predictive indicator (EPPI). We validate a robust performance of the EPPI risk scoring system in multiple independent data sets, comprising a total of 2,071 early-stage NSCLC tumors. Patients are stratified according to the universal cutoff score based on the EPPI when applied in the clinical setting; the low-risk group has significantly better survival outcome. The functional EPPI gene set represents a potential genomic tool to improve patient selection in early-stage NSCLC to further derive the best benefits of ACT and prevent unnecessary treatment or ACT-associated morbidity.Prognosis and prediction of adjuvant chemotherapy response in non-small cell lung cancer can have significant clinical impact. Here, the authors show that differential expression of a 29 extracellular matrix gene indicator, EPPI, can predict patient outcome.