Comparison of in vitro scratch wound assay experimental procedures

Abstract

Fibroblast migration is an important aspect of wound healing. Different factors can influence migration and as such proper wound healing. In vitro scratch wound assays are used to examine cellular migration. However, the wide array of techniques available reduces reproducibility of findings. In this paper, we compare two techniques for wound creation; i.e. the exclusion method or scratching of cell monolayers. Furthermore, we investigate if analysis software influences experimental outcome by comparing both commercially and freely available analysis software. Besides, we examine the effect of cortisol on migration behavior of fibroblasts and identify possible caveats in experimental design. Results show a significantly reduced migration of fibroblasts when wounds are created using a cell exclusion method. Furthermore, addition of cortisol to the cell culture media only reduced migration of fibroblast monolayers that had been scratched but not in those where wounds were created using the exclusion method. A possible explanation related to cytokine expression is discussed.

Keywords: Fibroblast; In vitro migration; Stress hormones; Wound healing.

Fig. 1

Comparison of wound after gap creation (A–C) and 24 h after (E–G). A, E: scratch created using IncuCyte Wound Maker Tool. B, F: Scratched cell monolayer by means of pipette tip. C, G: Open wound area created with Ibidi insert. D: comparison of wound width at baseline shows largest gap created using IncuCyte Wound Maker Tool followed by pipette scratch. Gaps created using Ibidi inserts had smallest distance and showed less variation. Scale bar: 200 μm. Graph shows mean values and standard deviation, IncuCyte: n = 43, pipette tip: n = 49, insert: n = 45. Arrow heads show examples of damaged cells at the wound edge.

Fig. 2

A) Reduced migration after 24 h of fibroblasts using exclusion method compared to scratching the monolayer. B&C) Cortisol delays migration capacity of scratched fibroblasts (pipette tip) but not for open wound areas created using the Ibidi insert (insert). *p < 0.05, ***p < 0.001 ***p < 0.001.

Fig. 3

Effect of cortisol (1 μmol/L) exposure on wound closure by NHDF cells evaluated over time as measured with the InuCyte sytem. Between 12 and 60 h a significant difference was observed between the two groups p < 0.001. n = 8 per group, repeated three times.

Fig. 4

Effect of software on data output. No significant differences were found between the groups. Graph shows mean values and standard deviation, n = 21.