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Stolwijk J.A.,Albany State University | Stolwijk J.A.,Applied BioPhysics Inc. | Renken C.W.,Applied BioPhysics Inc. | Trebak M.,Albany State University
Pflugers Archiv European Journal of Physiology | Year: 2015

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate. © 2014, Springer-Verlag Berlin Heidelberg.


Stolwijk J.A.,University of Regensburg | Hartmann C.,Universitaetsmedizin Mainz | Balani P.,Institute Of Bioengineering And Nanotechnology, Singapore | Albermann S.,Merck Serono GmbH | And 3 more authors.
Biosensors and Bioelectronics | Year: 2011

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90min. When membrane-impermeable, bioactive compounds like N 3 - or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS). © 2011 Elsevier B.V.


PubMed | University of Oregon and Applied BioPhysics Inc.
Type: | Journal: Stem cell research & therapy | Year: 2015

Regenerative medicine studies using autologous bone marrow mononuclear cells (BM-MNCs) have shown improved clinical outcomes that correlate to in vitro BM-MNC invasive capacity. The current Boyden-chamber assay for testing invasive capacity is labor-intensive, provides only a single time point, and takes 36 hours to collect data and results, which is not practical from a clinical cell delivery perspective. To develop a rapid, sensitive and reproducible invasion assay, we employed Electric Cell-substrate Impedance Sensing (ECIS) technology. Chemokine-directed BM-MNC cell invasion across a Matrigel-coated Transwell filter was measurable within minutes using the ECIS system we developed. This ECIS-Transwell chamber system provides a rapid and sensitive test of stem and progenitor cell invasive capacity for evaluation of stem cell functionality to provide timely clinical data for selection of patients likely to realize clinical benefit in regenerative medicine treatments. This device could also supply robust unambiguous, reproducible and cost effective data as a potency assay for cell product release and regulatory strategies.


Giaever I.,Applied BioPhysics Inc | Keese C.R.,Applied BioPhysics Inc
Cancer Metastasis - Biology and Treatment | Year: 2012

A personal account of the invention, growth and commercialization of Electric Cell-substrate Impedance Sensing (ECIS) by the inventors of the technology. From the first experiments at the General Electric Research and Development Center in the early 1980s to the outgrowth of applications and finally the incorporation of Applied BioPhysics, Inc., the chapter provides an historical and often amusing account of the evolution of ECIS. © Springer Science+Business Media Dordrecht 2012.


Patent
Applied Biophysics Inc. | Date: 2012-02-10

A filter device is provided which includes a well, at least one top access opening to the well, and a horizontally-disposed filter. The well, which includes an inner surface at least partially defining the well, also includes a first well chamber to accommodate a fluid and a second well chamber to accommodate a fluid. The first well chamber and the second well chamber are separate chambers, and the inner surface at least partially defines the first well chamber and at least partially defines the second well chamber. The at least one top access opening provides independent top access to the first well chamber and the second well chamber, and the horizontally-disposed filter is positioned between and at least partially separates the separate first well chamber and second well chamber of the well.


Ebrahimi C.M.,San Diego State University | Sheen T.R.,San Diego State University | Renken C.W.,Applied BioPhysics Inc | Gottlieb R.A.,San Diego State University | Doran K.S.,San Diego State University
Infection and Immunity | Year: 2011

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax disease in humans and animals. Systemic infection is characterized by septicemia, toxemia, and meningitis, the main neurological complication associated with high mortality. We have shown previously that B. anthracis Sterne is capable of blood-brain barrier (BBB) penetration, establishing the classic signs of meningitis, and that infection is dependent on the expression of both major anthrax toxins, lethal toxin (LT) and edema toxin (ET). Here we further investigate the contribution of the individual toxins to BBB disruption using isogenic toxin mutants deficient in lethal factor, ΔLF, and edema factor, ΔEF. Acute infection with B. anthracis Sterne and the ΔLF mutant resulted in disruption of human brain microvascular endothelial cell (hBMEC) monolayer integrity and tight junction protein zona occludens-1, while the result for cells infected with the ΔEF mutant was similar to that for the noninfected control. A significant decrease in bacterial invasion of BBB endothelium in vitro was observed during infection with the ΔLF strain, suggesting a prominent role for LT in promoting BBB interaction. Further, treatment of hBMECs with purified LT or chemicals that mimic LT action on host signaling pathways rescued the hypoinvasive phenotype of the ΔLF mutant and resulted in increased bacterial uptake. We also observed that toxin expression reduced bacterial intracellular survival by inducing the bulk degradative autophagy pathway in host cells. Finally, in a murine model of anthrax meningitis, mice infected with the ΔLF mutant exhibited no mortality, brain bacterial load, or evidence of meningitis compared to mice infected with the parental or ΔEF strains. © 2011, American Society for Microbiology.


Patent
Applied Biophysics Inc. | Date: 2012-07-23

Method and apparatus for processing a cell culture are provided. The method includes establishing a cell culture within a holding device having one or more wells, each well holding a cell culture, and including a well substrate with at least one electrode in contact with the cell culture; periodically applying at least one electrical pulse to the at least one electrode to prevent cells from attaching to and achieving confluence over the at least one electrode while allowing cells to attach to and achieve confluence over other portions of the well substrate; and discontinuing the periodically applying of the at least one electrical pulse to the at least one electrode after cells have achieved confluence over the other portions of the well substrate, and thereafter, monitoring the cell culture to monitor migration of cells over the electrode(s) from the other portions of the well substrate.


Patent
Applied Biophysics Inc. | Date: 2015-11-03

A filter device is provided which includes a well, at least one top access opening to the well, and a horizontally-disposed filter. The well, which includes an inner surface at least partially defining the well, also includes a first well chamber to accommodate a fluid and a second well chamber to accommodate a fluid. The first well chamber and the second well chamber are separate chambers, and the inner surface at least partially defines the first well chamber and at least partially defines the second well chamber. The at least one top access opening provides independent top access to the first well chamber and the second well chamber, and the horizontally-disposed filter is positioned between and at least partially separates the separate first well chamber and second well chamber of the well.


Trademark
Applied BioPhysics Inc. | Date: 2014-03-04

Graphic visual representations of multi-parametric, high content, phenotypic cell data through plotting of 2-D or 3-D coordinates.


Trademark
Applied BioPhysics Inc. | Date: 2010-12-14

Electric or electronic sensors for electrically sensing cell-substrate impedance changes for monitoring properties, morphological changes and behavior of cultured cells; sensor arrays for electrically sensing cell-substrate impedance changes for monitoring properties, morphological changes and behavior of cultured cells; cultureware, namely, customized disposable electrode array plate having multiple wells for electrically sensing cell-substrate impedance changes for monitoring properties, morphological changes and behavior of cultured cells; and scientific instruments, namely, cell analyzers in the nature of electric sensors for electrically sensing cell-substrate impedance changes for monitoring properties, morphological changes and behavior of cultured cells.

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