Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures.

Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression.

Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs.

Direct drug cocktail analyses using microscale vortex-assisted electroporation.

Combination therapy has become one of the leading approaches for treating complex diseases because it coadministers clinically proven drugs to concurrently target multiple signaling pathways of diseased cells. Identification of synergic drug combinations at their respective effective doses without unwanted accumulative side effects is the key to success for such therapy. In this work, we demonstrate the feasibility of the vortex-assisted microfluidic electroporation system for direct drug cocktail analyses where drug substances were individually delivered into cytosols in a sequential and dosage-controlled manner.

Microscale vortex-assisted electroporator for sequential molecular delivery.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system's ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability.

Improved diffuse fluorescence flow cytometer prototype for high sensitivity detection of rare circulating cells in vivo.

Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed "diffuse fluorescence flow cytometry" (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work.

p38 signaling and receptor recycling events in a microfluidic endothelial cell adhesion assay.

Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method.

Separation of two phenotypically similar cell types via a single common marker in microfluidic channels.

To isolate clinically and biologically relevant cell types from a heterogeneous population, fluorescent or magnetic tagging together with knowledge of surface biomarker profiles represents the state of the art. To date, it remains exceedingly difficult to separate phenotypically and physically similar cell types from a mixed population. We report a microfluidic platform engineered to separate two highly similar cell types using a single antibody by taking advantage of subtle variations in surface receptor density and cell size.

Lectin-functionalized microchannels for characterizing pluripotent cells and early differentiation.

Embryonic stem (ES) cells are capable of proliferating and differentiating to form cells of the three embryonic germ layers, namely, endoderm, mesoderm, and ectoderm. The utilization of human ES cell derivatives requires the ability to direct differentiation to specific lineages in defined, efficient, and scalable systems. Better markers are needed to identify early differentiation.

Instrument for fluorescence sensing of circulating cells with diffuse light in mice in vivo.

Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo.

Validation of a device for fluorescence sensing of rare circulating cells with diffusive light in an optical flow phantom model.

Detection and quantification of rare circulating cells in biological tissues is an important problem and has many applications in biomedical research. Current methods normally involve extraction of blood samples and counting of cells ex vivo, or the use of microscopy-based fluorescence in vivo flow cytometry. The goal of this work is to develop an instrument for non-invasively enumerating very rare circulating cells in small animals with diffuse light with several orders of magnitude sensitivity improvement versus current approaches.

Lectin-mediated microfluidic capture and release of leukemic lymphocytes from whole blood.

Lectins are a group of proteins that bind specifically and reversibly to mono- and oligosaccharide carbohydrate structures that are present on the surfaces of mammalian cells. The use of lectins as capture agents in microfluidic channels was examined with a focus on cells associated with T and B lymphocytic leukemia. In addition to examining the adhesion of Jurkat T and Raji B lymphocytes to a broad panel of lectins, this work also examined the capture of these cells from whole blood.

Receptor expression changes as a basis for endothelial cell identification using microfluidic channels.

Microfluidic channels functionalized with adhesive ligands are versatile platforms for cell separation in a variety of applications. However, not much is known about how the adhesiveness of targeted cell types can vary within such channels due to the combined influence of fluid shear forces and exposure to ligands. Using microfluidic channels and the tetrapeptide ligand arg-glu-asp-val (REDV), we demonstrate how such dynamic changes can provide a basis for the identification of three distinct phenotypes of endothelial cells: human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells (HMVECs), and endothelial colony forming cells (ECFCs).