microscale-vortex-assisted-electroporator-for-sequential-molecular

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 ...

Harvard University

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). This distinction is accomplished by characterizing changes in the adhesion profiles of the three cell types in REDV-coated microfluidic channels induced by soluble REDV and fluid shear forces. The significance of this technique lies in the ability to distinguish very similar cell-types without fluorescent label-based staining or flow cytometry.

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

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. Captured T and B lymphocytes were eluted from the microfluidic devices with a solution of the lectin's inhibiting sugar. The capture and release steps were accomplished in under 1 h. The significance of this work lies within the realm of low-cost capture of abundant target cells with non-stimulatory elution capability.

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

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. Lectins have been reported as an attractive alternative to the common stem cell markers. They have been used to identify, characterize, and isolate various cell subpopulations on the basis of the presentation of specific carbohydrate groups on the cell surface. This article demonstrates how simple adhesion assays in lectin-coated microfluidic channels can provide key information on the interaction of lectins with ES and definitive endoderm cells and thereby track early differentiation. The microfluidic approach incorporates both binding strength and cell surface receptor density, whereas traditional flow cytometry only incorporates the latter. Both approaches are examined and shown to be complementary with the microfluidic approach providing more biologically relevant information.

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

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. This platform utilizes antibody-conjugated surfaces in microfluidic channels together with precise modulation of fluid shear stresses to accomplish selective fractionation in a continuous flow process. Antibody conjugation density variation on the adhesive surfaces is achieved by covalently immobilizing an antibody in the presence of poly(ethylene glycol). This platform is used to demonstrate separation of two CD31 positive cell types, human umbilical vein endothelial cells and human micro vascular endothelial cells.

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

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. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs) is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV) and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome.

Dwayne A. L. Vickers

The Rowland Institute

Harvard University

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Dwayne A. L. Vickers

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Rowland Institute

Harvard University

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

The Rowland Institute