An effective device to enable consistent scratches for in vitro scratch assays

Abstract

Background: The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by mechanically removing cells through manual scraping with a sharp-edged tool. This step is traditionally done with pipette tips and is unsuitable for high-throughput assays, as the created scratches are highly variable in width and position. Commercially available solutions are often expensive, and require specific cultureware which might not be suitable for all studies.

Results: In this study, we have developed a flexible cell scratch device comprising a single wounding tool, a guide and an imaging template for consistent and reproducible scratch assays in 96-well plates. Our results showed that the device produced a more consistent scratch profile compared to the conventional method of using pipette tips. The imaging template also allowed operators to easily locate and image the same region of interest at different time points, which potentially could be used for other assays.

Conclusions: Our flexible yet effective scratch device thus enables robust scratch assays that can be applied to different experimental needs, providing researchers with an easy and reliable tool for their studies.

Keywords: Cell migration; Device; Prototype; Scratch assay; Wounding healing.

Fig. 1

Design of Scratch Setup. A Parts of wounding tool consisting of the top, spring, piston, pin, and base. B Pin tip can be fabricated with different diameters resulting in different scratch widths profile. C Assembly of wounding tool through the guide (slit panel) which guides the scratch process within each well of the 96-well plate. D Assembly of the 96-well plate on the imaging template consisting of reticle design for the monitoring of cell migration over the scratch area

Fig. 2

Scratch assay on HDF cell line. A Representative images of scratch profiles created by the scratch device (wounding tool and guide), 200 µL tip and 1000 µL tip. Black lines indicate sites where measurements were taken for analysis of scratch widths. B Scratch width measurements. Standard deviations were calculated from measurements across the scratch as shown in A. C Box and whisker plot indicating minimum, maximum, median, upper, and lower quartile values across the means of all wells. D Representative whole well image of scratches. Yellow lines go through the centre of the wells

Fig. 3

Scratch assay on HaCaT cell line. A Representative images of scratch profiles created by the scratch device (wounding tool and guide) and 1000 µL tip. B Scratch width measurements. Standard deviations were calculated from measurements across the scratch, as shown in Fig. 2A. C Box and whisker plot indicating minimum, maximum, median, upper, and lower quartile values across means of all wells. D Representative whole well image of scratches. Yellow lines go through the centre of the wells

Fig. 4

Use of the imaging template. Representative images of the wells with imaging template marking shown (yellow arrows) on Day 0 and Day 2. Black dotted lines indicate scratch borders on Day 0