A high throughput, interactive imaging, bright‐field wound healing assay

Abstract

The wound healing assay is a commonly used technique to measure cell motility and migration. Traditional methods of performing the wound healing assay suffer from low throughput and a lack of quantitative data analysis. We have developed a new method to perform a high-throughput wound healing assay that produces quantitative data using the LEAP™ instrument. The LEAP™ instrument is used to create reproducible wounds in each well of a 96-well plate by laser ablation. The LEAP™ then records bright field images of each well at several time points. A custom texture segmentation algorithm is used to determine the wound area of each well at each time point. This texture segmentation analysis can provide faster and more accurate image analysis than traditional methods. Experimental results show that reproducible wounds are created by laser ablation with a wound area that varies by less than 10%. This method was tested by confirming that neuregulin-2β increases the rate of wound healing by MCF7 cells in a dose dependent manner. This automated wound healing assay has greatly improved the speed and accuracy, making it a suitable high-throughput method for drug screening.

Advances in Wound-Healing Assays for Probing Collective Cell Migration

“…Furthermore, the optical and physical barrier assays with proper surface modification provide useful approaches to investigate the roles of ECM proteins in the collective migration process. 33,42 When cells are introduced to a free region without injury, such as those in vitro applications dealing with tumor invasion and tissue regeneration, a barrier assay may eliminate the effect of cell injury. 43 This method is also useful for those research topics concerning the surface coating effects on collective cell migration.…”

“…In this method, the cells in the predefined wound region were eliminated by laser ablation based on the photomechanical effect. 42 The motion of the laser pulse and the microscopy observation can be computer controlled, and thus this automated method is suitable for multiwell assays with high efficiency. Other advantages of optical wounding methods include the ability to create repeatable wounds, create wounds with arbitrary shapes, and perform automated microscopic observation using the optical interface.…”

Collective Cell Migration: A Mechanistic Perspective

“…This method elegantly overcomes most of the disadvantages of the wounding done with a needle and allows full control of the shape and size of the wound while leaving the substrate undamaged. However, it requires specialized and expensive equipment (77).…”

In vitro wound healing assays – state of the art

“…Pixels with surrounding areas having large pixel intensity variations (areas populated with cells) will appear bright, whereas areas with low pixel intensity variation (smooth areas) will appear dark. By applying a pixel threshold, the entropy image is converted into a binary image, which is inverted and further processed by removing noisy areas and smoothing out the surface of the wound area to create a continuous wound area to be quantified [20]. As a MATLAB ® script is used, this tool is fairly easy to implement.…”

“…Using laser pulses, cells were removed in a defined circular area. Data collection of bright-field microscopy images at certain time points and subsequent determination of the wound areas using customized algorithms (see also "Tools and algorithms for data analysis") were carried out for final data analysis [20]. Optical wounding offers a high throughput process under sterile conditions leading to reproducible injuries.…”