In Vitro Wounding Models Using the Electric Cell-Substrate Impedance Sensing (ECIS)-Zθ Technology.

Rebecca Johnson,Andrea Gu,Dan Kho,Simon O’Carroll,E Graham

doi:10.3390/bios8040090

Rebecca Johnson, Andrea Gu + Show 3 more

Open Access

https://doi.org/10.3390/bios8040090

Copy DOI

Journal: Biosensors	Publication Date: Oct 11, 2018
Citations: 10	License type: CC BY 4.0

Affiliation: University of Auckland

Abstract

Electric Cell-Substrate Impedance Sensing (ECIS) can produce reproducible wounding models by mechanically disrupting a cell monolayer. This study compared in vitro wound-healing using human cerebral microvascular endothelial cells (hCMVEC) with both single electrode (8W1E) and multiple electrodes (8W10E+) arrays. Measurements of hCMVEC migration and barrier functions were conducted, revealing variable levels of barrier disruption could be achieved by altering the duration and magnitude of the applied current. In all scenarios, the barrier (Rb) did not recover the strength observed prior to injury. Localization of junctional proteins following wounding were analyzed by immunocytochemistry. Following wounding, cell migration was generally faster on the 8W10E+ than the 8W1E array. Immunohistochemical analysis revealed non-viable cells remained on the 8W1E electrodes but not the 8W10E+ electrodes. However, viable cells partially remained on the 8W10E+ electrodes following wounding. In addition, the 8W10E+ electrodes demonstrated variation in cell loss across electrodes within the same well. This suggests the type of wounding is different on the two array types. However, our data show both arrays can be used to model incomplete barrier recovery and therefore both have potential for testing of drugs to improve endothelial barrier function. This is the first time that the possibility of using the 8W10E+ array as a wounding model is addressed. We highlight the differences in wounding produced between the two arrays, and can be used to study the underlying causes for impaired barrier function following CNS injuries.

Highlights

Compromised blood-brain barrier (BBB) function caused by neurological disease or injury leads to increased barrier permeability and infiltration of peripheral leukocytes into the CNS which can be detrimental [1,2]
The human cerebral microvascular endothelial cell line was purchased from Applied Biological Materials, and has been extensively characterized in terms of junctional protein expression and transendothelial electrical resistance (TEER) (O’Carroll et al, 2015; Wiltshire et al, 2016)
This study set out to establish whether it is feasible to develop a model of endothelial cell wounding using the Electric Cell-Substrate Impedance Sensing (ECIS) 8W10E+ array, in order to test treatments to strengthen an injured barrier in future studies

Summary

Introduction

Compromised blood-brain barrier (BBB) function caused by neurological disease or injury leads to increased barrier permeability and infiltration of peripheral leukocytes into the CNS which can be detrimental [1,2]. The ECIS-Zθ is an in vitro system that monitors real-time cellular behaviours and movements via gold film electrodes and a number of different plate configurations are available [4]. These electrodes are 4.9 × 104 μm and are connected to a larger counter electrode where a non-invasive alternating current flows with a frequency range of 10 to 105 Hz [5]. Changes in the current flow are measured as impedance (Z), which gives insight into two aspects of cellular behaviours and movements at different frequencies [6]

Methods

Results

Discussion

Conclusion