Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

Previously used in vitro or ex vivo models lack dermal cell components and complexity of skin tissue. This ex situ SCAD assay overcomes a set limitation and enables studying the development of scars outside of wounded animal. The SCAD assay allows visualization of fibroblast migration and scar formation in skin microenvironments.

It enables screening libraries of activators or inhibitors to understand the mechanistic basis of scar formation. High-throughput screening assays of flat libraries using the SCAD model in understanding fundamental process and the molecule course involved in wound repair. The SCAD assay is easy to perform using the skin explants.

For consistent scar, it is recommended to select post-natal day zero or day one skin. After sacrificing post-natal day zero or day one newborn pup, use a sterile surgical scalpel to carefully excise 1.5-by-1.5-centimeter full thickness dorsal back skin till the skeletal muscle layer. Peel the skin using sterile curved forceps, ensuring that the superficial fascia is intact with the underlying panniculus carnosus muscle.

Wash the excised tissue with 50 to 100 milliliters of cold DMEM F-12 medium to remove contaminating blood. Then, wash the tissue with Hanks balanced salt solution to maintain tissue and cell viability. Place the skin upside down with superficial fascia on top in a 10-centimeter Petri dish containing DMEM F-12 medium.

Then, using a disposable two-millimeter biopsy punch, excise full-thickness round skin pieces to generate scabbed tissues, ensuring that superficial fascia is intact with underlying panniculus carnosus muscle until the epidermis. Add 200 microliters of fresh DMEM F-12 complete medium without phenol red into each well of a 96-well plate. Using sterile forceps, transfer and fully submerge individual scabbed tissue upside down to the wells of a 96-well plate.

Transfer the plate to a cell culture incubator maintained under standard conditions. On days two and four of culture, remove the medium leaving 10 microliters in the well and add fresh pre-warmed DMEM F-12 complete medium along with the treatment compounds to maintain continued cell and tissue viability conditions. For live imaging of SCADs, prepare 30 milliliters of 2 to 3%low-melting agarose solution in PBS in a glass bottle by heating in a microwave.

After boiling, immediately transfer the bottle and cool the liquid agarose solution in a 40-degrees-Celsius water bath. Next, transfer the SCAD tissue with fascia or scar facing upward onto the center of a 35-millimeter dish. Then embed the SCAD at room temperature by slowly pouring 40-degrees-Celsius liquid agarose onto the tissue using a 1000-microliter pipette tip.

Agarose polymerizes within two minutes. After polymerization, add two milliliters of pre-warmed DMEM F-12 complete medium. Then using a confocal or a multiphoton microscope equipped with suitable incubation systems explained in the manuscript, acquire time-lapse images of day zero to day one.

For tissue harvesting, wash the tissues at relevant time points by replacing the media with sterile PBS. Using sterile forceps, transfer each SCAD to a 1.5-milliliter microcentrifuge tube containing 500 microliters of 2%paraformaldehyde to fix the tissues overnight at four degrees Celsius. The next day, wash the tissues three times with PBS before proceeding to two-or three-dimensional immunofluorescence staining as described in the text manuscript.

The representative whole-mount bright-field images of SCADs on days zero and five are shown here. The Masson's trichrome staining of vertical tissue sections reveals signatures of tissue scarring, tissue contraction, and accumulation of extracellular matrix at the scar core at days zero and five. The immunofluorescence staining of SCADs at days zero and five are shown.

From early and late stage scar, the ingrained positive fibroblast is shown in green and the ingrained negative fibroblast in red. For three-dimensional imaging, tissues were embedded in agarose solution and topped with PBSGT. The representative images demonstrate exclusive localization of N-cadherin protein at the scar site of a day-five SCAD.

The three-dimensional time lapse imaging setup equipped with an incubation chamber displayed early events of progression of the fibroblast swarms over the first 12 hours or early stages of scar development. The graphical representation of single-cell tracked cellular trajectories of individual fibroblasts over scar development is presented here. It's crucial to place the SCAD tissue upside down inside the well plate so the fascia faces upwards.

Failure to do this would result in unavoidable variations in migration patterns. This procedure allows to examine dermal layers for treatments or culture using chemical modulators, neutralizing antibodies, or viral methods. This helps in the assessment of pathological fibrotic responses across diverse medical settings.

We showed the expression of connexin-43 and N-cadherin as key molecules involved in fascia mobilization upon wounding. The SCAD methodology allows the identification of novel therapeutic strategies to curtail scarring and fibrosis.