Abstract
Here, we describe an in vitro epithelial wound-healing assay using Electric Cell-Substrate Impedance Sensing (ECIS) technology. The ECIS technology is a real time cell growth assay based on a small (250 μm diameter) active gold electrode which resistance is measured continuously. When intestinal epithelial cells reach confluency on the gold electrode, resistances reach a plateau. For the wound-healing assays, confluent intestinal epithelial monolayers are subjected to a current of 40 kHz frequency, 1,400 μA amplitude, and 30-second duration. This kills the cells around the small active gold electrode, causing detachment and generating a wound that is healed by surrounding cells that have not been submitted to the current pulse. Wound healing is then assessed by continuous resistance measurements for approximately 30 h after wound. Both cell wounding and measurements of the subsequent healing process are carried out under computer control that takes online measurements each 30 s and stores the data. ECIS technology can be used to study the underlying causes for impaired mucosal healing and to test the efficacy of drugs in mucosal healing.
Keywords: Electric Cell-Substrate Impedance Sensing, Wound-healing, Intestinal epithelial cell monolayers, Ulcerative colitis (UC), Caco2-BBE cells
Background
Damage and impairment of the intestinal surface barrier are observed in the course of various diseases including Ulcerative Colitis (UC). Such characteristics may result in an increased penetration and absorption of toxic and immunogenic factors into the body leading to inflammation, uncontrolled immune response, and disequilibrium of the homeostasis of the host. Wound-healing assays in tissue culture have been used for many years to estimate the migration and proliferation rates of different cells under various culture conditions. These assays involve using instruments such as razor blades to “wound” a confluent monolayer of cells. The main disadvantages of these assays include a lack of reproducibility and an inability to precisely quantify wound healing. Here, we describe the ECIS technology, a non-invasive technique which monitors live cells in situ and in real time, which is an ideal cell-based system for the screening of potential drugs capable of achieving higher mucosal healing rates that provide significant clinical benefit for Ulcerative Colitis patients.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
EV is a recipient of the Career Development Award from the Crohn’s and Colitis Foundation. This protocol was adapted from published work by Charrier et al. (2005), Zhang et al. (2016) and Xiao et al. (2018).
Competing interests
The authors declare no conflicts of interest within this work.
References
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