Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Summary

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Abstract

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the β3 subunit of voltage-gated calcium channels (Cavβ3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cavβ3-deficient mice (Cavβ3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cavβ3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cavβ3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cavβ3-deficient mice or cells might also be applicable for specific molecules other than Cavβ3.

Introduction

Skin wound healing starts immediately after the skin injury in order to restore the skin's integrity and to protect the organism from infections. The wound healing process goes through four overlapping phases; coagulation, inflammation, new tissue formation, and tissue remodeling¹. Cell migration is crucial during these phases. Inflammatory cells, immune cells, keratinocytes, endothelial cells, and fibroblasts are activated at different time points and invade the wound area². Methods to investigate wound healing in vitro and in vivo are of great interest not only to understand the underlying mechanisms but also to te....

Protocol

All experimental procedures were approved and performed in accordance with the ethics regulations and the animal welfare committees of Saarland and Saarland University.

1 Primary cell culture and siRNA transfection

NOTE: In the described method, primary fibroblasts are used. These cells play a crucial role in wound healing and tissue remodeling¹¹. In this experiment, the Cacnb3 gene, encoding the Cavβ3 subunit of high voltage-gated calcium channels¹² was down-regulated, thereby showing its role in cell migration in vitro and skin wound repair in vivo

Results

The scratch assay was performed on a confluent cell monolayer of wild-type and β3-deficient MEFs (Figure 1c). After performing the "scratch" using a 200 μL pipette tip, cells from both genotypes migrate into the scratch area and close the gap. Images were taken after 6, 10 and 30 h (Figure 1a). Cell migration was quantified as the percentage (%) of scratch area repopulated by migrating cells 6 hours after performing the scratch........

Discussion

In this manuscript, we describe an in vitro and in vivo wound healing assay and correlate the results obtained. For the in vitro assay, we used primary mouse fibroblasts^4,14,15 which play an important role in wound healing and tissue remodeling¹¹. Other adherent cell types growing as monolayers (e.g., epithelial cells, endothelial cells, keratinocytes) can be used as well. Plating the same number of viabl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9 % NaCl
1 ml syringes	BD Plastipak	303172
6 well plate	Corning	3516
Biopsy punch	Kai Industries	48201	2 mm
Cacnb3 Mouse siRNA Oligo Duplex (Locus ID 12297)	Origene	SR415626
Depilation cream			any depilation cream
Dexpanthenol 5% (BEPANTHEN)	Bayer	3400935940179.00	(BEPANTHEN)
Dihydroxylidinothiazine hydrochloride (Xylazine)	Bayer Health Care		Rompun 2%
Dulbecco's Modified Eagle Medium (DMEM)	Gibco by life technologies	41966-029
Fetal bovine serum	Gibco by life technologies	10270-106
Hexagon full nut
Ketamine hydrochloride	Zoetis		KETASET
Light microscope	Keyence, Osaka, Japan	BZ-8000	Similar microscopes might be used
Lipofectamine RNAiMAX Transfection Reagent	Thermo Fisher Scientific	13778075
Micro-forceps
Micro-Scissors
Mouse restrainer	Home-made
Normal scissors
Objective	Nikon	plan apo 10x/0.45
Opti-MEM	Gibco by life technologies	51985-026
Polypropylene sutures
Screwdriver
Skin disinfectant (octeniderm)	Schülke & Mayr GmbH	118212
Slotted cheese head screw
Snap ring
Snap ring plier
Surgical microscope with camera	Leica	Leica M651
Titanium frames for the skinfold chamber	IROLA	160001	Halteblech M
Wire piler