Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.
Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.
Chronic, non-healing wounds, which are highly prevalent in the elderly and diabetic, are a majorly unappreciated area of clinical unmet need. These wounds present a major physical and psychological burden to patients and cost healthcare providers billions each year to treat1. Despite improved understanding of wound biology and advancements in technology, up to 40% of chronic wounds still fail to heal following best standard care2. Thus, 14-26% of patients with diabetic foot ulcers subsequently require amputation3, while 5-year post-amputation mortality rate stands at approximately 70%4. As a result, there is an urgent requirement to develop efficacious new therapies to improve patient quality of life while reducing the substantial healthcare burden imposed by poor healing wounds. Poorly predictive pre-clinical models remain a significant hurdle to the development of effective new therapies.
Wound repair is a dynamic and multifaceted process involving a diverse range of cell types, countless levels of communication and a tissue environment that is temporally remodeled. Skin healing is underpinned by four major reparative stages: hemostasis, inflammation, proliferation, and matrix remodeling. These stages ultimately act to prevent blood loss and infection, close the wound surface (a process termed re-epithelialization) and return the skin to an uninjured state5. Chronic wounds are associated with diverse etiology and widespread perturbation to healing processes6, further complicating the identification of therapeutic targets. Nevertheless, a broad range of models have been developed to both elucidate the molecular and cellular drivers of wound pathology and test new therapeutic approaches7.
The most used wound repair model is acute wounding in the mouse. Mice are highly tractable for mechanistic studies and provide validated models of ageing and diabetes8. Despite the general similarities shown amongst mouse and human healing, between-species differences in skin structure and healing dynamics remain. This means most murine wound research does not easily translate to the clinic9. Consequently, there has been a push towards human in vitro and ex vivo systems with high applicability and translatability10,11.
Here we provide an in-depth protocol for performing partial thickness excisional wounds in ex vivo human skin. We also outline our whole-mount staining approach as a highly reproducible method of evaluating ex vivo human skin healing. We show the trajectory of epidermal repair (re-epithelialization) and subsequent barrier formation, evaluating the rate of wound closure in healthy versus diabetic human skin. Finally, we demonstrate how whole-mount staining can be adapted for use with a range of antibodies to assess various aspects of the healing response.
Human skin was obtained from patients undergoing reconstructive surgery at Castle Hill Hospital and Hull Royal Infirmary (Hull, UK) under full informed, written patient consent, institutional guidelines, and ethical approval (LRECs: 17/SC/0220 and 19/NE/0150). Non-diabetic skin was collected from patients undergoing routine surgery (mean age = 68). Diabetic skin was selected from donors who had established type II diabetes and a history of ulceration (mean age = 81). Samples from surgery were transported in holding media and processed immediately upon arrival at the laboratory. All experimental steps using unfixed human tissue were performed at Biosafety Level-2 (BSL-2) in a class II laminar flow biosafety cabinet.
1. Preparation of skin culture media and staining reagents
NOTE: All reagent and consumable details are provided in the Table of Materials. Ensure all reagents and equipment used for the processing and culture of human tissue are sterile. Sterilize instruments prior to the use and decontaminate with disinfectant following contact with the tissue. Decontaminate waste products in 1% disinfectant before disposal.
2. Preparation of skin for wounding
NOTE: These steps should be performed in a class II laminar flow biosafety cabinet.
3. Creating ex vivo human skin wounds
NOTE: These steps should be performed in a class II laminar flow biosafety cabinet.
4. Whole-mount staining of ex vivo wounds
NOTE: This section describes immunofluorescence and immunoperoxidase staining methods. Mix all reagents well before use.
5. Imaging and quantification
In this report, we present a novel ex vivo skin wounding and whole-mount staining approach to assess factors that influence the human skin repair response. Figure 1A shows a schematic of the procedural pipeline, which can be performed in 3-10 days, depending on wound incubation times. The partial thickness wounds are cultured on membrane stacks at the air : membrane interface and can be collected for whole-mount staining, embedded in paraffin or OCT medium for general histology, or frozen in liquid nitrogen for biochemical analysis (Figure 1B). We generally create 2 mm partial thickness wounds within the center of 6 mm explants. However, the size of the wound and surrounding explant may be altered depending on requirements. The whole-mount procedure has been successfully adapted for both immunoperoxidase and immunofluorescence staining methods (Figure 1C).
Immunofluorescence allows for the probing of tissue with multiple antibodies. For this, we advise using primary antibodies raised in different species, and species-matched fluorescently conjugated secondary antibodies to limit cross-species reactivity. Antibody concentrations and incubation times will need to be optimized. If background staining is observed, reduce antibody concentrations, increase wash steps, and add blocking buffer to the secondary antibody. Fresh tissue viability can be directly assessed with commercial viability dyes (see Table of Materials). We also show that tissue may be fixed post viability staining and successfully imaged when it is practically suitable (Figure 1D).
Figure 1: The human ex vivo wounding and whole-mount staining approach. (A) Pipeline depicting the procedural workflow from collecting skin and performing ex vivo wounding, to staining tissue and analyzing data. (B) Diagram demonstrating the human ex vivo skin wound culture system with analyses routinely performed on the tissue. (C) Whole-mount staining can be employed using both immunoperoxidase and immunofluorescence techniques. K14 = keratin 14. (D) Live tissue may be stained with commercial viability dyes and imaged successfully post fixation. Bar = 100 µm. This staining was performed in non-diabetic skin. Please click here to view a larger version of this figure.
The most widely applicable use for whole-mount staining of wounds is to determine wound closure rate in a more reproducible manner than can be provided via histological sectioning. Percentage closure was quantified as percentage re-epithelialization of the wound surface, as demonstrated in Figure 2A. Percentage area coverage of specific markers can be measured from the total wound area or as a percentage of the re-epithelialized wound. We characterized healing in healthy (non-diabetic) versus diabetic skin across a time course of seven days, collecting wounds at each day post-wounding (representative images, Figure 2B). Healthy skin wounds closed over time as expected, with full closure observed in most samples by day 4-5. On the contrary, diabetic skin wounds failed to close fully within the seven-day analysis period (Figure 2C). A significant delay in wound closure was observed between healthy and diabetic skin wounds when comparing healing rates at each time-point post-injury (P < 0.001 to day 6, P < 0.05 at day 6 and P < 0.05 to P < 0.001 at day 7).
Following assessment of overall wound closure rates; we measured the percentage of the entire wound area (outer area in Figure 2A) where K14 positive cells could be visualized (green staining in Figure 2B). Interestingly, we observed that in healthy ex vivo skin wounds, K14 staining peaked at day 2 and then rapidly declined (significance at each time-point versus the day 2 peak, Figure 2D). This is likely reflecting re-formation of the early epidermal barrier, excluding K14 antibody penetration through differentiated epidermal layers (see Figure 2E schematic). During the re-epithelialization process, basal layer (K14+ve) keratinocytes migrate inwards over the open wound such that the epidermis closer to the outer wound edge forms earlier than the epidermis closer to the inner wound edge (migrating front). While the front edge of the newly formed epidermis continues to migrate to close the remaining open wound, the outer edge epidermis begins to differentiate to reform the other epidermal layers. In early healing, we would thus expect to see most of the re-epithelialized area consists of basal (K14+ve) cells, while in later repair K14 staining is lost as the epidermis differentiates from the outside inwards (see whole-mount images in Figure 2E). Therefore, the decline in K14 staining shown in Figure 2D (downward arrows) correlates with increased epidermal differentiation. Interestingly, visible K14 staining peaked earlier in healthy (day 2) versus diabetic (day 4) wounds, further demonstrating that re-epithelialization and subsequent epidermal differentiation are delayed in diabetic skin wounds.
Figure 2: Whole-mount staining reveals perturbed healing rates in diabetic versus healthy skin. (A) The method used to quantify wound closure from outer and inner wound measurements. Brightfield images show keratin 14 (K14) staining in red. Bar = 300 µm. (B) Representative images of healing over time (day post-wounding) in healthy and diabetic skin. Bar = 500 µm. K14 = green. DAPI = blue nuclei. (C) Quantification of wound closure rates (percentage re-epithelialization) showing that ex vivo wounds from healthy skin close significantly faster than ex vivo wounds from diabetic skin. H = healthy. Db = diabetic. (D) Percentage K14 staining peaks earlier in healthy versus diabetic skin and then declines in line with increased epidermal differentiation (down arrows). (E) K14 (basal epidermal cell) staining is lost as the epidermis differentiates. D = differentiated. ND = not differentiated. White dotted lines depict inner and outer wound edges. White arrows = direction of migration. n = 6 wounds per donor, per time point. Mean +/- SEM. * = P < 0.05, ** = P < 0.01 and *** = P < 0.001. Healthy and diabetic compared at each healing time point in C (P value for least significant comparison). Temporal change in K14 staining compared to peak for each donor in D. Please click here to view a larger version of this figure.
We next used whole-mount staining to explore tissue expression and localization of other wound-relevant markers in non-diabetic skin (Figure 3). All antibodies used and their working concentrations are provided in the Table of Materials. Blood vessels in the open wound stained positively with alpha smooth muscle actin (a-SMA) antibody, used in combination with K14 to delineate the epidermal edges in lower power images (Figure 3A). The dermal matrix was stained with antibodies against collagen type I (COL 1) and fibronectin (Fn). Here collagen was observed as abundant thick fibers while fibronectin fibers were sparse, wavy, and thin (Figure 3A). Our whole-mount staining approach is also able to provide cell level resolution of staining, as demonstrated for K14-positive keratinocytes (Figure 3B).
Finally, we show that human ex vivo wounds possess resident immune cells, with Langerhans cells detected around newly formed epidermis at day 3 post-wounding (Figure 3C). Indeed, these results suggest that whole-mount staining may be used to investigate key features of the healing response including inflammation, proliferation, and the extracellular matrix (Figure 4A). Taken together, our data reveal that the combined ex vivo skin wounding and whole-mount staining procedure is a valid method to assess various aspects of healthy and diabetic (pathological) human skin repair.
Figure 3: Optimization of the whole-mount staining approach for use with other antibodies. (A) Blood vessels were stained with alpha smooth muscle actin (α-SMA, green) and keratin 14 (K14, red), while matrix fibers were stained with collagen I (COL 1, red) and fibronectin (Fn, green). (B) The whole-mount procedure provides up to cell level resolution of localization (K14, green; K1, red). (C) CD1a+ve Langerhans cells (green) observed in newly formed epidermis. DAPI = blue nuclei. Bar = 100 µm. White dotted lines show inner and outer wound edges and separate wound from epidermis. This staining was performed in non-diabetic skin. Please click here to view a larger version of this figure.
Figure 4: Validity of the whole-mount staining procedure for assessing wound healing. (A) Illustration depicting how the whole-mount staining technique can evaluate wound-relevant processes. Antibodies used = red text. K14 = keratin 14. COL 1 = collagen 1. Fn = fibronectin. (B) The whole-mount staining procedure (blue arrows) introduces less variability to wound closure measurements than standard histological analysis (red arrows). S1 = section 1. WE = wound edge. Bar = 300 µm. This staining was performed in non-diabetic skin. Please click here to view a larger version of this figure.
In this experimental protocol, we describe an optimized method for evaluating wound closure in human ex vivo skin using whole-mount tissue staining. This is an important resource to allow critical evaluation of potential wound treatments, and to provide better understanding of the human wound repair response. We have published healing assessment in ex vivo skin wounds previously12,13, yet in these reports the whole-mount staining approach was not used to measure wound closure. Whole-mount staining is far easier and requires less technical experience than standard histology, which involves paraffin or OCT embedding and sectioning of samples. The whole-mount procedure also reduces experimental variability, allowing quantification of the entire wound and not just a single transverse section at a defined position within the tissue (see Figure 4B for comparative illustration). We fully support the importance of quantifying healing of the entire non-symmetrical wound structure, as clearly outlined by Rhea and Dunnwald for murine acute wounds14. These authors showed the importance of serially sectioning in vivo excisional wounds for reproducible and precise measurements of wound morphology. Serial sectioning could equally be applied to human ex vivo wounds; however, for accurate quantification of wound closure and re-epithelialization, high throughput whole-mount staining should be the preferred method. We note that this whole-mount staining protocol should also be compatible with subsequent processing (wax or OCT) for traditional histological analysis.
Whole-mount staining is not without disadvantages. While it affords higher reproducibility in wound healing experiments, it does require the use of more tissue for analysis than standard histological techniques. This may be an issue where tissue access is limited, particularly where multiple antibodies need to be assessed. An alternative approach would be to employ an incisional wounding method where wound width is relatively uniform and variability is reduced (as shown in mouse and human wounds15,16). However, excisional wounds remain more applicable to most pathological wound types17.
In this study, 2 mm partial thickness wounds were created within the center of 6 mm skin explants. This method may be optimized for alternative excisional wound and explant sizes at different skin depths18. In addition, the force required to generate wounds will vary between donors, where aged skin will require less force to biopsy. We would also avoid using skin displaying prominent stretch marks or other structural alterations. We have validated a range of antibodies to consider different aspects of the ex vivo healing response. This protocol may also be used with other skin-relevant antibodies, where antibody concentrations and incubation times will need to be optimized. Nevertheless, we believe our protocol is most suited to absolute quantification of total wound closure, followed by spatial assessment of specific proteins of interest. While whole-mount provides reduced resolution of immunolocalization versus standard histological analysis of tissue sections, it provides additional 3D information that is missing from standard 2D histology.
One caveat of assessing healing in ex vivo skin versus in vivo models is that it lacks a systemic response. An important aspect of wound repair is inflammation and subsequent tissue granulation, which is caused by an influx of inflammatory cells and endothelial cells from the vasculature19. Despite this limitation, ex vivo skin still provides a better recapitulation of clinical healing than cell-based wound assays. In vitro experiments in general involve single cell type monolayers or co-cultures grown on tissue culture plastic, whereas ex vivo skin provides a native environment to explore cell behavior. More recently, a number of skin equivalent systems have emerged, where skin is grown in a laboratory setting from artificial matrix and isolated skin cells20,21. Although these models mimic human skin better than most in vitro approaches, they still do not fully simulate the native tissue environment and are generally too fragile to injure reproducibly. Additionally, we (and others) have demonstrated that ex vivo human skin tissue retains resident immune cells, which will no doubt contribute to repair22,23. Future work should now focus on extending the viability and immunocompetency of the ex vivo model for late-stage healing assessment24. One option is further advancement of promising organ-on-a-chip technologies capable of prolonging tissue viability and maintaining native skin architecture for up to two weeks in culture25. Ex vivo models have also begun to consider the importance of the skin inflammatory response by successfully incorporating immune cells, such as neutrophils, into the host tissue26 or injecting host tissue with antibodies to elicit an immune reaction27. We expect that these findings will pave the way for development of more refined and translatable methods in the future.
A major benefit of using ex vivo skin to measure wound closure is the ability to compare healing rates in healthy (e.g., non-diabetic) versus pathological (e.g., diabetic or aged) tissue. Here we showed that re-epithelialization and barrier formation are indeed impaired in diabetic versus healthy ex vivo wounds. Indeed, this provides a route for pre-clinical assessment of pathological repair, where ageing and diabetes are major risk factors for developing chronic wounds1. While in vitro pathological models exist, such as cells isolated from aged and diabetic tissue, or cells cultured in high glucose to mimic hyperglycemia28,29, these cells can quickly lose their phenotype once removed from the in vivo microenvironment. An important component of the extrinsic pathological healing environment is the dermal matrix, which is altered in both ageing and diabetes30. Indeed, this perturbed matrix affects the behavior of resident and naïve fibroblasts31,32. Thus, the importance of studying cells in their host tissue environment cannot be underestimated.
In summary, our protocol provides an important platform to quantify human wound re-epithelialization, explore regulatory factors and to test the validity and efficacy of potential therapeutics12,13. While pre-clinical testing does still require in vivo approaches, a combined strategy using ex vivo human tissue and in vivo murine wounding should refine the pre-clinical pathway, reducing animal use while increasing cross-species translatability.
The authors have nothing to disclose.
We would like to thank Mr Paolo Matteuci and Mr George Smith for providing patient tissue. We are also grateful to Miss Amber Rose Stafford for assisting with tissue collection and the Daisy Appeal for providing laboratory facilities.
50 mL Falcon Tubes | Falcon | 352070 | For skin washing |
1.5 ml TubeOne Microcentrifuge Tubes, Natural (Sterile) | Starlab | S1615-5510 | For whole-mount staining |
48-Well CytoOne Plate, TC-Treated | Starlab | CC7682-7548 | For whole-mount staining |
Acetic Acid Glacial | Fisher Chemical | A/0400/PB15 | Part of fixative |
Alkyltrimethylammonium Bromide | Sigma-Aldrich | M7635 | Part of fixative |
Anti-Alpha Smooth Muscle Actin Antibody [1A4] | Abcam | ab7817 | Stains blood vessels |
Anti-Collagen I Antibody | Abcam | ab34710 | Stains collagen |
Anti-Cytokeratin 14 Antibody [LL002] | Abcam | ab7800 | Stains epidermis |
CD1A Antibody (CTB6) | Santa Cruz Biotechnology | sc-5265 | Stains Langerhans cells |
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) | Thermo Fisher Scientific | 62247 | Counterstain for cell nuclei |
Falcon 60mm Petri dishes | Falcon | 353004 | Human ex vivo culture |
Fibronectin Antibody (EP5) | Santa Cruz Biotechnology | sc-8422 | Stains fibronectin |
Formaldehyde, Extra Pure, Solution 37-41%, SLR | Fisher Chemical | F/1501/PB17 | Part of fixative |
Gauze Swabs | Medisave | CS1650 | To clean skin |
Gibco™ Antibiotic-Antimycotic Solution | Thermo Fisher Scientific | 15240062 | Human ex vivo culture |
Gibco DMEM, high glucose, no glutamine | Thermo Fisher Scientific | 11960044 | Human ex vivo culture |
Gibco Fetal Bovine Serum | Thermo Fisher Scientific | 10500064 | Human ex vivo culture |
Gibco HBSS, no calcium, no magnesium | Thermo Fisher Scientific | 14170088 | Human ex vivo culture |
Gibco L-Glutamine (200 mM) | Thermo Fisher Scientific | 25030081 | Human ex vivo culture |
Hydrogen Peroxide | Sigma-Aldrich | H1009-100ML | For immunoperoxidase staining |
ImageJ Software | National Institutes of Health | N/A | For image analysis |
Invitrogen IgG (H+L) Cross-Adsorbed Goat anti-Mouse, Alexa Fluor 488 | Thermo Fisher Scientific | A11001 | Secondary antibody used depends on required fluorochromes and primary antibody |
Invitrogen IgG (H+L) Cross-Adsorbed Goat anti-Rabbit, Alexa Fluor 594 | Thermo Fisher Scientific | A11012 | Secondary antibody used depends on required fluorochromes and primary antibody |
Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells | Thermo Fisher Scientific | L3224 | For viability assessment of tissue |
Iris Forceps, 10 cm, Curved, 1×2 teeth | World Precision Instruments | 15917 | To create wounds |
Iris Scissors, 11 cm, Curved, SuperCut, Tungsten Carbide | World Precision Instruments | 501264 | To create wounds |
Iris Scissors, 11 cm, Straight, SuperCut, Tungsten Carbide | World Precision Instruments | 501263 | To remove adipose tissue |
Keratin 1 Polyclonal Antibody, Purified | Biolegend | 905201 | Stains epidermis |
Keratin 14 Polyclonal Antibody, Purified | Biolegend | 905301 | Stains epidermis |
LSM 710 Confocal Laser Scanning Microscope | Carl Zeiss | Discontinued | For fluorescent imaging |
Merck Millipore Absorbent pads | Merck Millipore | AP10045S0 | Human ex vivo culture |
Merck Millipore Nylon Hydrophilic Membrane Filters | Merck Millipore | HNWP04700 | Human ex vivo culture |
Normal Goat Serum Solution | Vector Laboratories | S-1000-20 | Animal serum used depends on secondary antibody |
Phosphate Buffer Solution | Sigma-Aldrich | P3619 | For wash buffer |
Sodium Azide | Sigma-Aldrich | S2002 | For blocking buffer |
Sodium Chloride | Fisher Bioreagents | BP358-212 | Part of fixative |
Sterilisation Pouches | Medisave | SH3710 | To sterilise instruments |
Stiefel 2mm biopsy punches | Medisave | BI0500 | For partial thickness wound |
Stiefel 6mm biopsy punches | Medisave | BI2000 | For outer explant |
Thermo Scientific Sterilin Standard 90mm Petri Dishes | Thermo Fisher Scientific | 101VR20 | To prepare skin |
Triton X-100 | Fisher Chemical | T/3751/08 | For wash buffer |
VECTASTAIN Elite ABC-HRP Kit, Peroxidase (Rabbit IgG) | Vector Laboratories | PK-6101 | For immunoperoxidase staining; HRP kit used depends on primary antibody |
Vector NovaRED Substrate Kit, Peroxidase (HRP) | Vector Laboratories | SK-4800 | For immunoperoxidase staining |
Wireless Digital Microscope | Jiusion | N/A | For brightfield imaging |