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.
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.
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 test new drugs and to develop new strategies aiming to ameliorate and accelerate skin wound healing.
To monitor and analyze cell migration, the scratch migration assay can be used. It is often referred to as in vitro wound healing assay. This method requires a cell culture facility3. It is a simple procedure, there is no need of high-end equipment and the assay can be performed in most cell biology laboratories. In this assay, a cell-free area is created by the mechanical disruption of a confluent cell monolayer, preferably epithelial- or endothelial-like cells or fibroblasts. Cells on the edge of the scratch will migrate in order to repopulate the created gap. Quantification of the decreasing cell-free area over time resembles the migration rate and indicates the time, which the cells need to close the gap. For this purpose, investigators can use either acutely isolated cells from WT mice or mice lacking a gene of interest4, or immortalized cells available from reliable cell repositories. The scratch assay allows studying the influence of pharmacologically active compounds or the effect of transfected cDNAs or siRNAs on cell migration.
In vivo, wound healing is a complex physiological process, requiring different cell types including keratinocytes, inflammatory cells, fibroblasts, immune cells and endothelial cells in order to restore the skin’s physical integrity as fast as possible1. Different methods to study in vivo wound healing have been developed and used in the past5,6,7,8. The dorsal skinfold chamber described in this article was previously used for wound healing assays9. It is used as a modified dorsal skinfold chamber preparation for mice. The modified skinfold chamber model has several advantages. 1) It minimizes skin contraction, which prevents observing the wound healing process and might influence wound repair in mice. 2) This chamber makes use of covering the wound with a glass coverslip, reducing tissue infections and desiccation, which could delay the healing process. 3) Blood flow and vascularization can be directly monitored. 4) It allows repetitive topical application of pharmacologically active compounds and reagents in order to treat the wound and accelerate healing9,10.
We identified the β3 subunit of high voltage-gated calcium channels (Cavβ3) as a potential target molecule to influence skin wound healing using two independent protocols, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. For the in vitro assay, we used primary fibroblasts, these cells do express the Cacnb3 gene encoding the Cavβ3 protein but lack depolarization-induced Ca2+ influx or voltage-dependent Ca2+ currents. We described a novel function of Cavβ3 in these fibroblasts: Cavβ3 binds to the inositol 1,4,5-trisphosphate receptor (IP3R) and constraints calcium release from the endoplasmic reticulum. Deletion of the Cacnb3 gene in mice leads to increased sensitivity of the IP3R for IP3, enhanced cell migration and increased skin wound repair4.
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 vivo4.
2. In vitro wound healing assay (scratch migration assay)
3. Analysis of the scratch area
4. In vivo skin wound healing assay
NOTE: C57BL/6 wild-type males (8-12 weeks old with 22-26 g body weight) and Cavβ3-deficient mice as a control are used for this study.
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. Migrating Cavβ3-deficient MEFs closed the scratch area significantly earlier than MEFs from wild-type mice (Figure 1a,b). To exclude any effect of cell proliferation, the scratch migration assay was performed in the presence of either 10% or 1% FCS. At 10% FCS both processes are present, the cell proliferation and migration, whereas at 1% FCS cell proliferation is minimized. Fibroblasts in 10% (Figure 1b, left) or 1% FCS (Figure 1b, right) showed a similar migration pattern, ruling out the possibility of cell proliferation contribution to the Cavβ3 observed phenotype. Cavβ3-deficient MEFs closed the gap significantly earlier than wild-type MEFs under both conditions. To confirm the Cavβ3-dependent effect observed in β3-deficient fibroblasts, wild-type fibroblasts were transfected with siRNA to down-regulate the Cavβ3 protein (Figure 1e). As a control for the down-regulation, immunoblots were performed to confirm the efficiency of the siRNA treatment. Two independent Cacnb3 specific siRNAs (siRNA1 and siRNA2), and a scrambled siRNA (as a control) were used. Fibroblasts treated with the Cacnb3-specific siRNAs behave like β3-deficient fibroblasts (Figure 1d), i.e., the migration is increased in the absence of Cavβ3 protein (Figure 1e).
In vivo, the dorsal skinfold chamber was implanted (Figure 2a,b) and a defined circular wound of 2 mm diameter was generated on the shaved back (Figure 2b) of the wild-type and Cavβ3-deficient mice (8 animals per genotype, 8-12 weeks old and 22-26 g weight). The wound was performed by removing the complete skin with epidermis and dermis. To compare skin wound healing between both genotypes, the wound area in the skin fold chamber was photographed directly after wounding (day 0) and then pictures were taken 3, 6, 10, and 14 days post wounding (Figure 2c). The sizes of the wounds were measured on these digital images and the wound area at a given day was expressed as the percentage (%) of the initial wound area (Figure 2d). Wound closure is increased in β3-deficient mice compared to wild-type controls. In contrast to the wild-type, the wound in β3-deficient mice was almost completely closed already after 10 days. At day 14 post-wounding, the wounds were completely closed in both genotypes (Figure 2c,d).
Figure 1: In vitro scratch migration assay. (a) Representative images of cultures from wild-type (WT, left) and Cavβ3 KO (β3 KO, right) primary mouse embryonic fibroblasts (MEFs) immediately, 6, 10 and 30 h after performing a scratch. Images were converted into 8-bit gray scale, and the contrast, as well as brightness, were adapted to maximally visualize the cell free area. Analysis of the cell free area (% of scratch area repopulated by migrating cells) was performed on the original 24-bit RGB images. (b) Bar graphs showing percentages (%) of scratch area repopulated by migrating cells after 6 hours either in the presence of high (10 %, left) or low (1%, right) fetal bovine serum (FCS) in wild-type (black) and Cavβ3 KO (red) experiments (c) Immunoblot: Protein extracts from wild-type brain (50 μg per lane) and fibroblasts (MEFs, 100 μg per lane) using a Cavβ3 specific antibody. The β3 protein (55 kDa) is present in wild-type brain (used as a control), and fibroblasts, but is absent in protein extracts of Cavβ3-deficient brain and fibroblasts prepared from Cavβ3-deficient mice. (d) Summary of the percentage of scratch area repopulated by migrating cells after 6 hours in wild-type cells treated with either scrambled siRNA (control, black) or two independent Cacnb3 siRNAs (siRNA1 and siRNA2, red open bars). (e) The corresponding immunoblot from the experiment in (d). Data are shown as mean ± SEM, p values were calculated using unpaired two-tailed Student’s t-test. Panels a and b have been modified from [Belkacemi et al., 2018]4. Please click here to view a larger version of this figure.
Figure 2: In vivo skin wound healing in mice. (a) Titanium frame interior side view showing one half of the titanium chamber and front side view showing the assembled titanium chamber with two symmetrical halves attached with screws and nuts. (b) Mouse after shaving the dorsal skin and mounting the dorsal skinfold chamber composed of two symmetric titanium frames (the weight of the titanium frame is around 2 g) and applying a circular wound (2 mm diameter). (c) Images of the wound directly after wounding (day 0) and 3, 6, 10, and 14 days post-wounding. The continuous process of wound closure, with complete epithelialization, is shown over 14 days in wild-type (WT, top) and Cavβ3 KO (β3 KO, bottom) mice. (d) At the time points indicated, the wound area was determined using a computer-assisted image analysis program and plotted as a percentage of the wound area immediately after injury at day 0 (mean ± SEM of n = 8, β3 KO mice and the corresponding wild-type control mice). P values were calculated using two-way ANOVA and Bonferroni’s multiple comparisons test. Panels c and d have been modified from [Belkacemi et al., 2018]4. Please click here to view a larger version of this figure.
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 fibroblasts4,14,15 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 viable and healthy cells and applying the scratch at the same degree of confluency is of paramount importance in order to obtain accurate and reproducible results. It is highly recommended to perform biological and technical replicates. In the present method, 6-well plates were used, but 12- or 24-well plates can be also used especially when cells are available only at limited numbers. In the case of siRNA treatment, immunoblot analysis after each set of experiments is mandatory to make sure that the target protein is efficiently down-regulated. Transfection reagent and time window should be tested and selected for each cell type before starting with the migration assay. In the case of fibroblasts and the Cacnb3 gene, it took 3 to 4 days to reach the desired level of down-regulation. In contrast, the scratch migration assay needs shorter times (6 h to 24 h). To avoid high variability in the scratch size, it is mandatory that the same investigator applies the scratch for each set of experiments, that equal pressure is administered by the pipette tip and to keep the wound as much as possible vertical to the marked line at the bottom of the plate across the cell monolayer. Application of a mechanical scratch across the cell monolayer leads to the release of different cellular factors (e.g., ATP) from damaged cells into the extracellular space. These factors would induce paracrine signaling including Ca2+-signaling in the neighboring cells, which in turn would influence cellular responses16. To avoid these effects, culture inserts can be used for plating cells and after removal of these inserts a cell-free gap is created without damaging the neighboring cells17. For high-throughput screening, the investigators might consider using instruments available on the market to create reproducible and consistent scratches in 96-well plates. To follow cell migration kinetics continuously over time, users can also consider using high-end commercial systems for automatic image capture. However, automated systems for scratch application and image capture are not always available because of high costs. A more accessible and cost-effective solution for time-lapse imaging would be for example using the system (ATLIS: an affordable time-lapse imaging and incubation system) described by Hernandez Vera and colleagues18.
In the absence of any cell proliferation inhibitors, repopulation of the gap in the scratch migration assay is a combination of cell migration and cell proliferation. To monitor only cell migration, cell proliferation can be suppressed for example by treatment with either Actinomycin C19 or Mitomycin C20. Adequate concentrations of these compounds must be carefully determined and tested to avoid the toxic effects of these compounds, which might affect the viability of the cells and their ability to migrate. As described in the present article, serum starvation or the reduction of the serum concentration in the culture medium is another way to reduce the effects of cell proliferation. Serum starvation is used in several other cell-based assays. It can induce a high number of cellular responses, which might interfere with the obtained results and interpretations21. Serum starvation must be carefully applied and its effect on the cell viability should be assessed before starting the experiment. In the present article, migration of primary fibroblasts in the presence of either 10% or 1% serum is shown (see Figure 1b). Migration rate, as expected, is slower at low concentrations of serum. However, β3-deficient fibroblasts migrate faster than wild-type cells at both conditions; low and high serum concentration.
The skin wound healing assay using the dorsal skinfold chamber is a relatively straightforward procedure to investigate skin wound closure over time in vivo. The implantation of the titanium dorsal skinfold chamber was described for the first time in rats22. Sorg et al. used this technique in SKH1-hr hairless mice to follow wound healing and formation of new blood vessels9. The skinfold chamber model described in this article has many advantages over the classical wound healing assays performed on the dorsal skin, on the ear23 or hind limb7 of mice. Covering the wound area with a glass coverslip prevents infections and tissue trauma and limits desiccation of the wound. The observation chamber covered with the glass coverslip can be opened at any time during the healing process, allowing topical application of different pharmacologically active compounds (e.g., siRNA for Cavβ3 as solutions or ointments) and the chamber can be closed again. Murine skin wounds healing process is composed of both contraction and epithelialization24. Using the dorsal skinfold chamber in mice minimizes skin contraction and gives the opportunity to study mainly the epithelialization process. It provides also a clear window to observe and monitor the wound closure process. One disadvantage of the titanium chamber is that the mouse has to carry the titanium chamber, which has a weight of around 2 g (7.7% of the weight of a 26 g mouse), for 14 days. This might cause some discomfort for the mouse, although it seems that mice tolerate well this chamber and they are comfortable and can easily reach food and water. The skin wound healing model presented in this article can study only one wound per mouse. Other published methods25,26 suggest applying two wounds per mouse which would reduce the number of animals needed for a study. It is of great importance to create a circular wound of similar size for all mice to get objective information as well as reliable and reproducible results. To take images of the wounds over time, mice were fixed on a mouse restrainer, placed on a stage, and the skinfold chamber is positioned under a stereomicroscope. Using the restrainer helps in avoiding anesthesia and minimizing stress for the mice. Mice can be sacrificed and tissues from the wounded area can be explanted and collected at different stages of the healing process (either after complete healing or at earlier time points) for histological analysis, RNA collection or protein biochemistry.
In summary, we have shown two techniques, an in vitro scratch assay using primary fibroblasts and an in vivo skin wound healing assay in mice. In both assays, wound healing/gap closure is increased in the absence of the Cavβ3 subunit of voltage-gated calcium channels. As with wild-type and Cavβ3-deficient mice or cells, both assays might well correlate in the absence or presence of other specific molecules.
The authors have nothing to disclose.
We thank Dr. Petra Weissgerber and the Transgene Unit of the SPF animal facility (project P2 of SFB 894) of the Medical Faculty and the animal facility at the Institute of Clinical and Experimental Surgery at the Medical Faculty of Saarland University, Homburg. We thank Dr. Andreas Beck for critical reading of the manuscript. This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich (SFB) 894, project A3 to A.B. and V.F.).
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 |