In this article, we present an optimized ex vivo human skin wounding protocol that can provide important efficacy data as part of a clinical translational pipeline. The main advantage of this technique is that it allows a high throughput accurate assessment of wound repair in living human skin. Demonstrating the procedure will be Elizabeth Roberts, a postdoctoral researcher, and Alexandria Kidd, a research assistant from our laboratory.
To prepare a skin tissue sample for wounding, place the skin dermis side down in a 90 millimeter Petri dish and use sterile scissors to remove the adipose tissue. When all the fat has been removed, place the skin in 25 milliliters of HBSS supplemented with antibiotics for 10 minutes at room temperature with intermittent shaking to remove any residual blood in adipose tissue. After 10 minutes, rinse the skin in 25 milliliters of DPBS.
After 10 minutes, repeat the HBSS rinse without antibiotics, and then perform a final rinse in 25 milliliters of DPBS. Then place a sterile nylon filter membrane onto the absorbent pad stack and dry the dermal side of the skin on sterile gauze in a 90 millimeter Petri dish to remove residual DPBS. Place the dried skin dermis side down on a clean 90 millimeter Petri dish lid to remove any residual DPBS and dab the epidermis dry with fresh sterile gauze.
To create a wound, press a two millimeter biopsy punch against the skin while holding the skin tight and twisting gently. Using a curved toothed tissue forceps, pick up each side of the approximately two millimeter diameter wound and hook curved Iris scissors under the wound. Use a six millimeter biopsy punch to biopsy around the central two millimeter wound to create a six millimeter explant with a partial thickness two millimeter wound in the center.
Then place the wound explant epidermis side up on the nylon filter membrane stack for a one to seven-day incubation at 32 to 37 degrees Celsius and 5%carbon dioxide. For fluorescent staining, collect the wound explants in 1.5 milliliter microcentrifuge tubes containing 500 microliters of skin fixative per tube and incubate the explants overnight at four degrees Celsius. The next day, replace the fixative with one milliliter of staining wash buffer.
After the wash, cover each sample with approximately 150 microliters of blocking buffer and incubate for one hour at room temperature. After removing the blocking buffer, add 150 microliters of primary antibody diluted in blocking buffer per tube and incubate the wound explants overnight at four degrees Celsius. The next morning, rinse the samples with 500 microliters of staining wash buffer containing 0.2%sodium azide followed by three rinses in regular wash buffer.
After the wash, add 150 microliters of an appropriate florescence-conjugated secondary antibody diluted in staining wash buffer to each well for a one-hour incubation at room temperature. At the end of the incubation, rinse the explant three times for 30 minutes in 500 microliters of staining wash buffer per wash before counter-staining each explant with 150 microliters of DAPI for 10 minutes at room temperature. Finally, wash the explant two times for 30 minutes with 500 microliters of washing buffer per wash.
For explant imaging, place a 60 millimeter Petri dish onto the imaging platform of a confocal microscope and add an approximately one milliliter layer of DPBS to the dish. Use forceps to transfer the wound explants to the dish. After setting up the imaging software as appropriate for the experiment, position the wound in the center of the imaging plane and acquire images of the wound biopsies.
To perform wireless digital microscope imaging to obtain high-quality images in a cost-effective manner, connect a phone or laptop to a wireless digital microscope. Place the explants wound side up onto a piece of lab tissue and remove any residual wash buffer from the sample. Then position the explant in the center of the field of view of the microscope and acquire images using the connected camera.
Immunoperoxidase and immunofluorescence can be used for whole mount tissue staining. In addition, live/dead staining can be performed on fresh tissue and imaged pre or post-fixation. Whole mount staining of wounds can be used to determine wound closure rates in a reproducible manner.
In this representative analysis, healthy skin wound closure was observed after four to five days while diabetic skin wounds failed to close fully within the seven-day analysis period. K14 expression peaked on day two in healthy skin wounds before rapidly declining with increased epidermal differentiation. This re-epithelialization and subsequent epidermal differentiation was delayed in the diabetic skin wounds.
The reformation of the early epidermal barrier excludes K14 antibody penetration within the differentiated epidermal layers. Early in the re-epithelialization process, K14 positive basal layer 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. Later in the repair stage, K14 staining is lost as the epidermis differentiates from the outside inwards.
Whole mount staining can also be used to study the expression and localization of other wound relevant markers in skin, as well as the presence of immune cells of interest at the wound site at different stages of healing. Generating reproducible excisional wounds can be challenging at first. We would advise practicing the wounding procedure using surplus skin.
