28 research outputs found
Marking 100 years since Rudolf Höber’s discovery of the insulating envelope surrounding cells and of the beta-dispersion exhibited by tissue
© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Electrical Bioimpedance 3 (2012): 74-79, doi:10.5617/jeb.401.Between 1910 and 1913 Rudolf Höber presented proof that the interiors of red blood cells and muscle cells contain conducting electrolytes, and that each conducting core is contained within an insulating membrane. He did this by demonstrating, in a series of remarkable electrical experiments, that the conductivity of compacted cell samples at low frequencies (~150 Hz) was about ten-times less than the value obtained at ~5 MHz. On perforation of the membrane, the low-frequency conductivity increased to a value approaching that exhibited at MHz frequencies. Apart from representing a major milestone in the development of cell biology and electrophysiology, Höber’s work was the first description of what we now call the dielectric b-dispersion exhibited by cell suspensions and fresh tissue
Spatial manipulation of cells and organelles using single electrode dielectrophoresis
Author Posting. © Author(s), 2012. This article is posted here by permission of Informa Healthcare USA, for personal use, not for redistribution. The definitive version was published in BioTechniques 52 (2012): 39-43, doi:10.2144/000113802.The selection, isolation, and accurate positioning of single cells in three dimensions are increasingly desirable in many areas of cell biology and tissue engineering. We describe the application of a simple and low cost dielectrophoretic device for picking out and relocating single target cells. The device consists of a single metal electrode and an AC signal generator. It does not require microfabrication technologies or sophisticated electronics. The dielectrophoretic manipulator also discriminates between live and dead cells and is capable of redistributing intracellular organelles.This research was funded by NIH-NCRR grant P41 RR001395 and supported by The Eugene and Millicent Bell Fellowship Fund in Tissue Engineering, the Hermann Foundation Research Development Fund Award, the Dennis and Alix Robinson Memorial, and the MBL Bell Center, grant GM092374.2012-07-0
Dielectrophoresis: A Review of Applications for Stem Cell Research
Dielectrophoresis can discriminate distinct cellular identities in heterogeneous populations, and monitor cell state changes associated with activation and clonal expansion, apoptosis, and necrosis, without the need for biochemical labels. Demonstrated capabilities include the enrichment of haematopoetic stem cells from bone marrow and peripheral blood, and adult stem cells from adipose tissue. Recent research suggests that this technique can predict the ultimate fate of neural stem cells after differentiation before the appearance of specific cell-surface proteins. This review summarises the properties of cells that contribute to their dielectrophoretic behaviour, and their relevance to stem cell research and translational applications
Intracellular potassium under osmotic stress determines the dielectrophoresis cross-over frequency of murine myeloma cells in the MHz range
Dielectrophoresis based discrimination of human embryonic stem cells from differentiating derivatives
Assessment of the dielectrophoresis(DEP) cross-over frequency (f xo), cell diameter, and derivative membranecapacitance (C m) values for a group of undifferentiated human embryonic stem cell (hESC) lines (H1, H9, RCM1, RH1), and for a transgenic subclone of H1 (T8) revealed that hESC lines could not be discriminated on their mean f xo and C m values, the latter of which ranged from 14 to 20 mF/m2. Differentiation of H1 and H9 to a mesenchymal stem cell-like phenotype resulted in similar significant increases in mean C m values to 41–49 mF/m2 in both lines (p < 0.0001). BMP4-induced differentiation of RCM1 to a trophoblast cell-like phenotype also resulted in a distinct and significant increase in mean C m value to 28 mF/m2 (p < 0.0001). The progressive transition to a higher membranecapacitance was also evident after each passage of cell culture as H9 cells transitioned to a mesenchymal stem cell-like state induced by growth on a substrate of hyaluronan. These findings confirm the existence of distinctive parameters between undifferentiated and differentiating cells on which future application of dielectrophoresis in the context of hESC manufacturing can be based
Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy.
Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account
Label- and amplification-free electrochemical detection of bacterial ribosomal RNA
Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40 min at room temperature without wash steps
Protein Dielectrophoresis: A Tale of Two Clausius-Mossottis—Or Something Else?
Standard DEP theory, based on the Clausius–Mossotti (CM) factor derived from solving the boundary-value problem of macroscopic electrostatics, fails to describe the dielectrophoresis (DEP) data obtained for 22 different globular proteins over the past three decades. The calculated DEP force appears far too small to overcome the dispersive forces associated with Brownian motion. An empirical theory, employing the equivalent of a molecular version of the macroscopic CM-factor, predicts a protein’s DEP response from the magnitude of the dielectric β-dispersion produced by its relaxing permanent dipole moment. A new theory, supported by molecular dynamics simulations, replaces the macroscopic boundary-value problem with calculation of the cross-correlation between the protein and water dipoles of its hydration shell. The empirical and formal theory predicts a positive DEP response for protein molecules up to MHz frequencies, a result consistently reported by electrode-based (eDEP) experiments. However, insulator-based (iDEP) experiments have reported negative DEP responses. This could result from crystallization or aggregation of the proteins (for which standard DEP theory predicts negative DEP) or the dominating influences of electrothermal and other electrokinetic (some non-linear) forces now being considered in iDEP theory