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Endothelial Barrier Toxicity Assay

Ncardia’s Endothelial Barrier Toxicity Assay offers a highly relevant, sensitive and predictive in vitro safety assessment of drug candidates using functional human endothelial cells, derived from induced pluripotent stem cells.

The assay includes high-content, real-time and label-free impedance-based technology (ECISTM) that allows detailed understanding of compound effects on vascular barrier function (which is deteriorated in drug-induced vascular leakage) by monitoring changes in electrical impedance across the endothelial cell monolayer. Our advanced data analysis capabilities enable calculation of time course changes in the barrier function of the cell layer (Rb; permeability), the degree of constricted flow of current under the cells (Alpha; cell-matrix interaction) and the cell membrane capacitance (Cap; a measure of cell viability). Such analysis enable discrimination between necrosis and apoptosis, between subtle loss of cell-cell contacts or acute cell death, or the detection of a subtle loss of cell-cell contacts which cannot be assessed when using overall resistance.

 

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Case study: Endothelial Barrier Toxicity Assay can capture drug induced vascular leakage by chemotherapeutics.

Vascular leakage is a dose-limiting adverse effect for many therapeutics under development and should be taken into account. This study investigated the effect of 2 test compounds in a concentration range over 21 days with the Endothelial Barrier Toxicity Assay.

 

Case study specifications:

Cell type

Ncardia human iPSC-derived endothelial cells

Format

96 wells
Technology High-content, real-time and label-free impedance-based technology (ECISTM)

Assay window

Long-term assessment of compound effects: 21 days

Assay controls

0.1% DMSO (negative control) on day 7-11

Test compounds

DNA alkylating cytotoxic agent
Tubulin inhibitor

Compound concentrations

1 pM, 10 pM, 100 pM, 1nM, 10 nM, 100 nM, 1 µM

Readout Barrier resistance (at 4 kHz)

hiPSC-derived Endothelial Cells form a tight barrier that remains stable in long-term culture (A), which resembles the quiescent status of healthy endothelium in the human body. Hence, the Endothelial Barrier Toxicity Assay enables assessment of compound effects on the endothelial barrier permeability. For instance, the endothelial barrier function of hiPSC-derived endothelial cells was challenged with two model drugs for cancer treatment: a DNA alkylating cytotoxic agent and a tubulin inhibitor that blocks cell devision (B and C, respectively). Upon treatment with these drugs, relevant and distinctive side effects on endothelial barrier functioning are defined: high concentrations of the DNA alkylating agent completely and irreversibly deteriorated barrier function, while the tubulin inhibitor treatment caused a transient opening of the barrier that could be restored after drug removal. Altogether, these results show that the Endothelial Barrier Toxicity Assay offers a highly relevant and suitable in vitro assay in early drug development for differential screening of drug-induced vascular leakage.