Cluster-Wells: A Technology for Routine and Rapid Isolation of Extremely Rare Circulating Tumor Cell Clusters from Unprocessed Whole Blood.

Isolation of extremely rare circulating tumor cell (CTC) clusters from the bloodstream of patients enables minimally invasive diagnosis and prognosis while providing information on their role in metastasis. A few technologies specifically developed for the enrichment of CTC clusters fail to achieve a high enough processing throughput to be practical in clinical settings or risk damaging large clusters owing to their structural design producing high shear forces. Here, we outline a methodology developed for rapid and effective enrichment of CTC clusters from cancer patients, independent of the cluster size and cell surface markers. Minimally invasive access to tumor cells in hematogenous circulation will be an integral part of cancer screening and personalized medicine.

High throughput, label-free isolation of circulating tumor cell clusters in meshed microwells.

Extremely rare circulating tumor cell (CTC) clusters are both increasingly appreciated as highly metastatic precursors and virtually unexplored. Technologies are primarily designed to detect single CTCs and often fail to account for the fragility of clusters or to leverage cluster-specific markers for higher sensitivity. Meanwhile, the few technologies targeting CTC clusters lack scalability. Here, we introduce the Cluster-Wells, which combines the speed and practicality of membrane filtration with the sensitive and deterministic screening afforded by microfluidic chips. The >100,000 microwells in the Cluster-Wells physically arrest CTC clusters in unprocessed whole blood, gently isolating virtually all clusters at a throughput of >25 mL/h, and allow viable clusters to be retrieved from the device. Using the Cluster-Wells, we isolated CTC clusters ranging from 2 to 100+ cells from prostate and ovarian cancer patients and analyzed a subset using RNA sequencing. Routine isolation of CTC clusters will democratize research on their utility in managing cancer.

Automated lightless cytometry on a microchip with adaptive immunomagnetic manipulation.

Surface expression of cell populations are often sought as diagnostic and prognostic biomarkers in hematology-oncology and infectious diseases, making flow cytometry an invaluable technique for both clinical and basic research applications. On the other hand, the reliance of flow cytometry on manual input parameters and user protocols for operation introduces variation between analyses while potentially leading to errors in measurements. In this work, we introduce an integrated flow cytometry microchip that automatically adapts to the sample it interrogates. Our device measures the antigen expression in a sample by automatically analyzing the response of immunomagnetically labeled cells to an external magnetic field through integrated electrical sensors and by continuously modulating the time the cells are subjected to the field for optimal sensitivity and dynamic range. Furthermore, the lack of optical illumination and fluorescence detectors enables automated analysis to be carried on a fully integrated platform that is particularly well suited for translation into point-of-care testing and mobile screening. We applied our automated cytometry chip on both pure and mixed cell populations and validated its operation by benchmarking against a conventional flow cytometer. By transforming the utility-proven flow cytometry, a technique that has long been dependent on an operator in centralized laboratories, into a standardized disposable test for bedside or home testing, the automated flow cytometry microchip introduced here has the potential to enable self-screening for telemedicine and wellness.

Electronic measurement of cell antigen expression in whole blood.

Membrane antigens are phenotypic signatures of cells used for distinguishing various subpopulations and, therefore, are of great interest for diagnosis of diseases and monitoring of patients in hematology and oncology. Existing methods to measure antigen expression of a target subpopulation in blood samples require labor-intensive lysis of contaminating cells and subsequent analysis with complex and bulky instruments in specialized laboratories. To address this long-standing limitation in clinical cytometry, we introduce a microchip-based technique that can directly measure surface expression of target cells in hematological samples. Our microchip isolates an immunomagnetically-labeled target cell population from the contaminating background in whole blood and then utilizes the differential responses of target cells to on-chip magnetic manipulation to estimate their antigen expression. Moreover, manipulating cells with chip-sized permanent magnets and performing quantitative measurements via an on-chip electrical sensor network allows the assay to be performed in a portable platform with no reliance on laboratory infrastructure. Using our technique, we could successfully measure expressions of the CD45 antigen that is commonly expressed by white blood cells, as well as CD34 that is expressed by scarce hematopoietic progenitor cells, which constitutes only ∼0.0001% of all blood cells, directly from whole blood. With our technology, flow cytometry can potentially become a rapid bedside or at-home testing method that is available around the clock in environments where this invaluable assay with proven clinical utility is currently either outsourced or not even accessible.

Capillary flow control in lateral flow assays via delaminating timers.

Lateral flow assays (LFAs) use capillary flow of liquids for simple detection of analytes. While useful for spontaneously wicking samples, the capillary flow inherently limits performing complex reactions that require timely application of multiple solutions. Here, we introduce a technique to control capillary flow on paper by imprinting roadblocks on the flow path with water-insoluble ink and using the gradual formation of a void between a wetted paper and a sheath polymer tape to create timers. Timers are drawn at strategic nodes to hold the capillary flow for a desired period and thereby enable multiple liquids to be introduced into multistep chemical reactions following a programmed sequence. Using our technique, we developed (i) an LFA with built-in signal amplification to detect human chorionic gonadotropin with an order of magnitude higher sensitivity than the conventional assay and (ii) a device to extract DNA from bodily fluids without relying on laboratory instruments.

Negative enrichment of circulating tumor cells from unmanipulated whole blood with a 3D printed device.

Reliable and routine isolation of circulating tumor cells (CTCs) from peripheral blood would allow effective monitoring of the disease and guide the development of personalized treatments. Negative enrichment of CTCs by depleting normal blood cells ensures against a biased selection of a subpopulation and allows the assay to be applied on different tumor types. Here, we report an additively manufactured microfluidic device that can negatively enrich viable CTCs from clinically-relevant volumes of unmanipulated whole blood samples. Our device depletes nucleated blood cells based on their surface antigens and the smaller anucleated cells based on their size. Enriched CTCs are made available off the device in suspension making our technique compatible with standard immunocytochemical, molecular and functional assays. Our device could achieve a ~ 2.34-log depletion by capturing > 99.5% of white blood cells from 10 mL of whole blood while recovering > 90% of spiked tumor cells. Furthermore, we demonstrated the capability of the device to isolate CTCs from blood samples collected from patients (n = 15) with prostate and pancreatic cancers in a pilot study. A universal CTC assay that can differentiate tumor cells from normal blood cells with the specificity of clinically established membrane antigens yet require no label has the potential to enable routine blood-based tumor biopsies at the point-of-care.

Circulating Tumor Cell Enrichment Technologies.

Circulating tumor cells (CTCs) are responsible for the metastatic spread of cancer and therefore are extremely valuable not only for basic research on cancer metastasis but also as potential biomarkers in diagnosing and managing cancer in the clinic. While relatively non-invasive access to the blood tissue presents an opportunity, CTCs are mixed with approximately billion-times more-populated blood cells in circulation. Therefore, the accuracy of technologies for reliable enrichment of the rare CTC population from blood samples is critical to the success of downstream analyses. The focus of this chapter is to provide the reader an overview of significant advances made in the development of diverse CTC enrichment technologies by presenting the strengths of individual techniques in addition to specific challenges remaining to be addressed.

Hybrid negative enrichment of circulating tumor cells from whole blood in a 3D-printed monolithic device.

Isolation and analysis of circulating tumor cells (CTCs) from blood samples present exciting opportunities for basic cancer research and personalized treatment of the disease. While microchip-based negative CTC enrichment offers both sensitive microfluidic cell screening and unbiased selection, conventional microchips are inherently limited by their capacity to deplete a large number of normal blood cells. In this paper, we use 3D printing to create a monolithic device that combines immunoaffinity-based microfluidic cell capture and a commercial membrane filter for negative enrichment of CTCs directly from whole blood. In our device, stacked layers of chemically-functionalized microfluidic channels capture millions of white blood cells (WBCs) in parallel without getting saturated and the leuko-depleted blood is post-filtered with a 3 µm-pore size membrane filter to eliminate anucleated blood cells. This hybrid negative enrichment approach facilitated direct extraction of viable CTCs off the chip on a membrane filter for downstream analysis. Immunofluorescence imaging of enriched cells showed ∼90% tumor cell recovery rate from simulated samples spiked with prostate, breast or ovarian cancer cells. We also demonstrated the feasibility of our approach for processing clinical samples by isolating prostate cancer CTCs directly from a 10 mL whole blood sample.

Electronic profiling of membrane antigen expression via immunomagnetic cell manipulation.

Membrane antigens control cell function by regulating biochemical interactions and hence are routinely used as diagnostic and prognostic targets in biomedicine. Fluorescent labeling and subsequent optical interrogation of cell membrane antigens, while highly effective, limit expression profiling to centralized facilities that can afford and operate complex instrumentation. Here, we introduce a cytometry technique that computes surface expression of immunomagnetically labeled cells by electrically tracking their trajectory under a magnetic field gradient on a microfluidic chip with a throughput of >500 cells per min. In addition to enabling the creation of a frugal cytometry platform, this immunomagnetic cell manipulation-based measurement approach allows direct expression profiling of target subpopulations from non-purified samples. We applied our technology to measure epithelial cell adhesion molecule expression on human breast cancer cells. Once calibrated, surface expression and size measurements match remarkably well with fluorescence-based measurements from a commercial flow cytometer. Quantitative measurements of biochemical and biophysical cell characteristics with a disposable cytometer have the potential to impact point of care testing of clinical samples particularly in resource limited settings.

Design and modeling of electrode networks for code-division multiplexed resistive pulse sensing in microfluidic devices.

A typical microfluidic device sorts, captures or fractionates sample constituents by exposing them to discriminating microenvironments. Direct electronic acquisition of such manipulation by a network of integrated sensors can provide a fast, integrated readout, replacing otherwise required microscopy. We have recently introduced a sensor technology, Microfluidic CODES, which allows us to multiplex resistive pulse sensors on a microfluidic device. Microfluidic CODES employs a network of micromachined coplanar electrodes such that particles passing over these electrodes produce distinguishable code sequences. In this paper, we explain the design process to specifically generate an orthogonal digital code set for an efficient and accurate demultiplexing of the sensor signals. We also introduce an equivalent circuit model for a network of code-multiplexed resistive pulse sensors by utilizing the Foster-Schwan model and conformal mapping, to model dynamic cell-electrode interaction in a non-uniform electric field. Our results closely match with both experimental measurements using cell lines and finite element analysis. The coding and modeling framework presented here will enable the design of code-division multiplexed resistive pulse sensors optimized to produce desired waveform patterns to ensure reliable and efficient decoding.