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

Summary

This protocol describes the generation of a skin-fascia explant termed “SCar like tissue in A Dish” or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

Abstract

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or "SCar-like tissue in A Dish" that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids researchers in understanding fundamental processes and mechanisms of wound repair, directly exploring the effects of modulators on wound healing outcomes.

Introduction

Wound healing is a process of restoration of breached wounds. Tissue injuries in invertebrates result in partial or complete regeneration. In contrast, mammals respond to deep injury by scarring, a process tailored to quickly seal wounds with dense plugs of matrix fibers that minimize the breached area and at the same time permanently deform the injured site^1,2,3. Large skin burns or deep open wounds in mammals result in pathological phenotypes such as hypertrophic or keloid scars^4,5. These exuberant scars cause a tremendous burden on clinical and global healthcare systems. In the US alone, scar management costs about $10 billion annually^6,7. Therefore, the development of relevant methodologies are required to better understand the fundemental processes and mechanisms involved in scar formation.

In recent years, a wide range of studies in mice has revealed heterogeneous fibroblast populations with distinct functional potencies based on their origins in certain skin locations^8,9,10. In back skin, Rinkevich et al., 2015, identified that a specific fibroblast population with an early embryonic expression of Engrailed-1 (En1), termed EPF (Engrailed positive fibroblast) contributes to cutaneous scarring upon wounding. Conversely,  another fibroblast lineage with no history of engrailed expression, Engrailed negative fibroblast (ENF), does not contribute to scar formation⁸. Fate mapping of these En1 lineages using Cre-driven transgenic mouse lines crossed to fluorescence reporter mouse lines such as R26^mTmG (En1Cre x R26^mTmG) allows visualization of EPF and ENF populations.

Studying fibroblast migration in vivo over several days is limited by ethical and technical constraints. Furthermore, compound, viral and neutralizing antibody library screens to modulate pathways involved in scarring is technically challenging. Previously used in vitro or ex vivo models lack the ability to visualize fibroblast migration and scar formation in genuine skin microenvironments, uniformity in scar development, as well as tissue complexity that emulates in vivo skin environments^11,12. To overcome the above limitations, we developed an ex vivo scarring assay termed SCAD (SCar-like tissue in A Dish)^13,14. This simple assay can be performed by excising 2 mm full-thickness skin containing the epidermis, the dermis, and the subcutaneous fascia regions and culturing them in serum-supplemented DMSO media for up to 5 days. Scars generated from SCAD reliably replicate transcriptomic and proteomic hallmarks of in vivo scars. In addition, SCADs generated from relevant transgenic mouse lines (e.g., En1 mice) crossed with fluorescent reporter mouse lines allow the visualization of fibroblast migration dynamics and scar development at an unprecedented resolution. Furthermore, this model can be easily adapted for any high throughput applications (e.g., compound library, antibody library, or viral screening)^13,14. In this article, we describe an optimized protocol to generate SCADs and subsequent downstream processing applications to study cellular and matrix dynamics in scar development.

Protocol

The model presented below provides a detailed step-by-step description of the generation of SCAD assay as briefly described in Jiang et al., 2020¹³. SCAD sample preparations were performed after sacrificing the animals as per the international and the Government of Upper Bavaria guidelines. Animals were housed at the animal facility of the Helmholtz Centre Munich. The rooms were maintained with optimal humidity and constant temperature with a 12 h light cycle. Animals were supplied with food and water ad libitum.

1. SCAD tissue preparation

1. Sacrifice newborn pups onpostnatal day 0 or day 1 (P0 or P1).
2. Carefully excise at least 1.5 cm x 1.5 cm full-thickness dorsal back skin until the skeletal muscle layer using a sterile surgical scalpel.
3. Peel the skin using sterile curved forceps, ensuring that the superficial fascia is intact with the underlying panniculus carnosus muscle.
4. Wash the excised tissue with 50-100 mL of cold DMEM/F-12 media to remove contaminating blood.
5. Wash once with Hanks' Balanced Salt Solution (HBSS) to maintain tissue and cell viability.
6. Place the skin upside down (superficial fascia on top) in a 10 cm Petri dish containing DMEM/F12 media.
7. Using a disposable 2 mm biopsy punch, excise full-thickness round skin pieces, ensuring that superficial fascia is intact with underlying panniculus carnosus muscle until the epidermis to generate SCAD tissues.
8. Prepare and fill 200 µL of DMEM/F12 (without phenol red) complete media-DMEM/F12 media supplemented with 10% FBS, 1x GlutaMAX, 1x MEM non-essential amino acids, and 1x Penicillin/streptomycin into individual wells of a 96-well plate.
9. Using sterile forceps, carefully transfer and fully submerge individual SCAD tissue upside down (fascia facing up) to the wells of a 96-well plate.
10. Transfer the plate to a cell culture incubator maintained under standard conditions (37 °C, 21%(v/v) oxygen, 5% (v/v) C0₂, and 95% humidity).

2. SCAD – Tissue culture

1. Culture SCADs in an incubator for up to 5 days.
2. On Day 2 and Day 4 of culture, replace the media with 200μl of fresh pre-warmed DMEM/F12 complete media to ensure continued cell and tissue viability conditions. Replace treatment compounds during each media change.
   NOTE: Ensure to leave 10 µL of media in the well to avoid tissues sticking to the wells.
3. Prepare SCADs for the following experiments: Live imaging (section 3), and Tissue Harvesting and 2D/3D immunofluorescence staining (section 4).

3. Live imaging of SCADs

1. Prepare a minimum of 30 mL of 2%-3% (w/v) low melting agarose solution in PBS in a glass bottle by heating it in a microwave until boiling.
2. Immediately transfer the bottle and cool the liquid agarose solution in a 40 °C water bath.
3. Transfer the SCAD tissue with fascia/Scar facing upward onto the center of a 35 mm dish.
4. Embed the SCAD at room temperature (RT) by slowly transferring 40 °C liquid agarose onto the 35 mm dish using a Pasteur pipette. Agarose polymerizes within 2 min.
5. Add 2 mL of pre-warmed DMEM/F12 complete media (without phenol red indicator).
6. Acquire time-lapse Images of day 0 SCADs up to 48 h using a confocal or a multiphoton microscope equipped with a suitable incubation system set to 37 °C, 21% (v/v) oxygen, 5% (v/v) C0₂, and 95% humidity.

4. Tissue harvesting and 2D/3D immunofluorescence staining

1. Wash the tissues at relevant time points by replacing the media with sterile PBS.
2. Using sterile forceps, carefully transfer individual SCADs to 1.5 mL microcentrifuge tubes containing 500 µL of 2% paraformaldehyde to fix the tissues overnight at 4 °C.
3. Wash the tissues three times with PBS and proceed with 2D/3D immunofluorescence staining.
4. 3D immunofluorescence staining
   1. Permeabilize the tissues by incubating in a 1.5 mL microcentrifuge tube containing 500 µL of PBS supplemented with 0.2% gelatin, 0.5% Triton-X100, and 0.01% Thimerosal (PBSGT) at RT for 24 h.
   2. Prepare an adequate amount of primary antibodies as per the manufacturer´s instructions and incubate SCAD tissues in 150 µL of PBSGT solution for 24 h at RT.
      NOTE: If the optimal antibody concentration for immune fluorescence is not reported by the manufacturer, prior antibody titration needs to be performed on control tissues using varying concentrations (e.g., 1:50, 1:200, 1:500, 1:1000).
   3. Gently wash the SCADs three times with PBS to remove the unbound primary antibody.
   4. Incubate tissue with 1:1000 relevant Alexa Fluor secondary antibodies in PBS overnight at RT.
   5. Gently wash the tissue for 30 min in PBS three times to remove excess unbound secondary antibodies.
   6. Transfer the tissue onto a 35 mm glass-bottom dish for confocal or multiphoton imaging.
      NOTE: When using water immersion objectives, embed the SCADs in 2 % low melting point agarose to immobilize the tissue to prevent drifting during image acquisition
5. 2D immunofluorescence staining
   1. Transfer the tissue to a cryo-mold and gently fill with optimal cutting temperature (OCT) compound to completely immerse the tissue.
   2. Gently adjust the orientation of the tissue, ensuring the absence of air bubbles to obtain a cross-section or a vertical section.
   3. Place the mold on dry ice for 20-30 min and incubate the block at -80 °C overnight.
   4. Set the blade temperature to -25 °C and specimen block temperature to -17 °C. Prepare 6 µm cryo-section using a cryostat, transfer the sections onto an adhesion slide, and store the slides in -20 °C freezer.
   5. Rinse the slides three times in PBS and incubate the slides in 5% Bovine Serum Albumin (BSA) w/v in PBST (PBS supplemented with 0.05% Tween) for 1 h.
   6. Add an appropriate amount of primary antibody to 150μl PGST, and incubate overnight at 4 °C
      NOTE: If the optimal antibody concentration for immune fluorescence is not reported by the manufacturer, prior antibody titration needs to be performed on control sections using varying concentrations (e.g., 1:50, 1:200, 1:500, 1:1000).
   7. Gently wash the sections three times with PBS to remove the unbound primary antibody.
   8. Incubate tissue with 1:1000 relevant Alexa Fluor secondary antibodies in PBS overnight at RT for 2 h.
   9. Gently wash the tissue for 5 min in PBS three times to remove excess unbound secondary antibodies.
   10. Mount the slides with a mounting medium containing DAPI and dry the slides in the dark at RT.
   11. Image the sections using a fluorescence microscope.

Representative Results

Generation of SCADs can be separated into three essential steps: Harvesting back skin from P0-P1 mice, generating of full-thickness biopsy punches, and subsequent culture of individual scads up to 5 days in 96-well plates. As a readout, this assay can further be applied to analyze the spatial and temporal aspects of scarring. The spatial analysis utilizes 2D and 3D immune-labeling of tissues to study spatial localization of cellular and matrix components within developing scar tissue. Spatiotemporal studies allow visualization of these ex-situ scars using tissue intrinsic or extrinsic fluorescent proteins that can be visualized using relevant 3D time-lapse imaging modalities.

The pivotal steps in this assay are setting up the experiment by carefully generating full-thickness biopsy punches, ensuring the presence of a fascial "jelly" layer, and culturing the tissue fully submerged upside down (epidermis facing the bottom of the 96-well plates). This allows the development of uniform scars, comparable migration dynamics of relevant fibroblast populations, and skin contraction across SCADs (Figure 1A). The versatility of SCAD assay allows harvesting the tissue across the entire spectrum of scar development. For example, if the aim of the experiment is to study the early dynamics of scarring, spatial and spatiotemporal analysis can be limited to D1 or D2 of SCAD culture (Figure 1B).

Representative whole-mount bright-field images of SCADs at day 0 and day 5 are shown in Figure 2A and Figure 2A', respectively. For 2D analysis, it is essential to embed the SCADs in OCT in perpendicular orientation devoid of air bubbles to ensure intactness of tissue after sectioning. These tissue sections can further be subjected to histological analysis (e.g., Masson trichrome staining-Figure 2B,2B') or Immunofluorescence staining (Figure 2C,2C') on the cryosections from En1^Cre,R26^mTmG mouse dorsal dermis; EPF in green and ENF in red from early (Figure 2D) and late-stage scar (Figure 2D').

3D and 3D time-lapse imaging of scars requires detecting fluorescence signals deep inside the tissue (400-800 µm). Excitation in near-infrared wavelengths using two-photon excitation enables such measurements without the need for tissue clearing, a methodology commonly used in 3D imaging to make tissue transparent by minimizing light scattering and absorption^15,16,17. We used an upright Multiphoton microscope equipped with a 25x water-dipping objective in combination with a tunable laser. Tissues were subjected to 3D immune staining using relevant probes. For 3D imaging, the tissue was embedded in a 2% low melting agarose solution to immobilize or prevent drift in tissue (Figure 3A) topped with PBSGT to match the refractive index required for the water immersion objective. Representative image in Figure 3B and Figure 3B' shows exclusive localization of N-Cadherin protein (pink/magenta-Alexa fluor 647) at the scar site of a day 5 SCAD from a En1^Cre,R26^mTmG back skin; EPF in green, and ENF in red. For 3D time-lapse imaging, a slight modification was made to the above setup by embedding the tissue in a 2% low melting agarose solution topped with DMEM/F12 media without phenol red indicator instead of PBS. To ensure right conditions for "living" SCAD tissue during imaging, a suitable incubation chamber was added so that all phenotypes are correctly recorded during imaging (Figure 3C). Representative snapshots of early fibroblast swarms are shown in Figure 3D, Figure 3D' and Figure 3D'' over the first 12 h/early stages of scar development.

Figure 1: Schematic workflow of SCAD assay. (A) Back skin from postnatal day 0 / day 1 pups are excised, ensuring the tissue integrity containing the following dermal layers: Epidermis (E), papillary and reticular dermis (PD and RD), and Fascia layer (F) followed by 2 mm biopsy punches to obtain day 0 SCADs. (B) SCADs can then be subsequently cultured for up to 5 days with an optional intermittent harvest/tissue fixation step based on the nature of individual experiments (dotted arrows) with a culture media change step on day 2 and day 4. Please click here to view a larger version of this figure.

Figure 2: 2D immunofluorescence staining. Representative 2D readouts to study early and late-stage scars using (A and A') Bright field whole-mount image at day 0 (A) and day 5 (A'). (B and B') Masson's trichrome staining of vertical tissue sections to reveal signatures of tissue scarring-tissue contraction and accumulation of ECM at the scar core (yellow filled triangle) at day 0 (B) and day 5 (B'). (C and C') Immunofluorescence analysis of day 0 (C) and day 5 (C') SCADs generated from En1^Cre,R26^mTmG back skin.EPFs are shown in green, ENF in red, and DAPI in blue from early (D) and late stage (D´) scar. Please click here to view a larger version of this figure.

Figure 3: Schematic setup and representative readouts of SCADs for 3D spatio and spatiotemporal analysis. (A) Schematic representation of embedding the SCADs in 2% low melting temperature agarose gel under an upright fluorescent microscope and (B) representative image of a 3D immunofluorescent stained day 5 SCAD generated from En1^Cre,R26^mTmG mouse dermis and immune-stained with N-Cadherin antibody (Magenta).EPFs are shown in green and ENFs are shown in red. (C) Schematic representation 3D imaging SCAD setup equipped with an incubation chamber: 37 °C, 21% (v/v) oxygen, 5% (v/v) C0₂, and 95% humidity. (D) Representative results of live imaging showing early events of progression of fibroblast swarms (white dashed circle) in the first 12 h of day 0 and day 1 stage SCAD. (E) Graphical representation of single-cell tracked cellular trajectories of individual fibroblasts at D, D' and D''. Please click here to view a larger version of this figure.

Discussion

Several models have already been developed to understand scar formation after injury. While a lot of advances have been rendered in this regard but, actual mechanisms are still not clear. In contrast to the previous technique, the SCAD model incorporates all cell types and cutaneous layers, thereby maintaining the complexity of native skin^18,19. This methodology is capable of generating fundamental datasets that are important in understanding molecular mechanisms that drive scar formation. The assay is designed in a way that is simple, minimalistic, yet capable of producing fundamental and functional readouts that are crucial for understanding fibroblast migration, ECM deposition, epidermal/muscle contraction^13,14. It is relatively less complex than in vivo setups, and deviations with respect to the sample can be minimized. Further, this assay can be easily coupled with high throughput analysis pipelines. Wherein the entire spectrum or libraries of inhibitors or activators such as chemical compounds, neutralizing antibodies, siRNA, or viral methodologies that reduce or promote scarring could be applied with relative ease to minimal amounts of excised tissues. Regarding the limitation of this ex vivo scar model, this assay cannot be applied to study phenotypic or dynamic aspects of a) non-resident Immune swarms in scar development b) vascularization c) scar maturation as tissue is not viable beyond 5 days post-injury.

Before biopsy punches excision, it is important that there must be no residual blood remaining in the tissue as it might interfere with the proceedings of the experiment. We recommend washing the tissue more than once with HBSS if necessary, to make sure of any residual blood from the tissue is eliminated. Also, changing media on alternate days needs to be performed with special care, as,  the dermal and epidermal layers may separate due to the small size of the tissue. Therefore, it is imperative to practice the procedure properly in advance before starting actual experiments. To avoid repeated separation of the tissue layers, we suggest that during media change, 10 µl of residual media can be kept before replacing with fresh media in order to stabilize the tissue and minimize the change in surface tension on the tissue caused by the media added to the well.

For 2D analysis pipelines, we recommend equilibrating the tissue with liquid optimal cutting temperature compound at room temperature for a few minutes before mounting the tissue in a block. This prevents tissue separation due to ice crystal formation during sectioning. 3D/3D time-lapse imaging pipelines should be adequately modified based on the available microscope modality. For an upright microscope (with or without water immersion objective), embedding the tissue in agarose or using superglue prevents the physical shift of tissue during imaging. Water immersion objectives allow 3D/3D time-lapse imaging using PBS or colorless media (without phenol red). For an inverted microscope, tissue could be placed on a glass-bottom/imaging plate and immobilized using a drop of low melting temperature agarose and topped with suitable media to ensure viability during live imaging. In both modalities, a compatible incubation system may be used to ensure the right temperature, humidity, and gas supply during imaging.

In conclusion, the SCAD model enables the identification and characterization of novel molecular cues and mechanistic pathways involved in mammalian wound healing^13,14. The protocol provides a detailed description of the generation of SCADs and potential downstream applications and analysis to provide insight into scar development.

Materials

10% Tween 20, Nonionic Detergent	Biorad Laboratories	1610781
Bovine serum albumin, Cold ethanol fract	Sigma	A4503-50G
DMEM/F-12, HEPES, no phenol red-500 mL	LIFE Technologies	11039021
DPBS, no calcium, no magnesium	Gibco	14190169
Epredia Cryostar NX70 Cryostat	Thermo Scientific
Epredia SuperFrost Plus Adhesion slides	Fisher scientific	J1800AMNZ	Adhesion slides
Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin-500 mL	LIFE Technologies	10500064
Fluoromount-G with DAPI	Life Technologies	00 4959 52	Mounting medium with DAPI
Forceps curved with fine points with guidepinstainless steel(tweezers)125 mm length	Fisher Scientific	12381369
Gelatin from porcine skin	Sigma	G2500-100G
GlutaMAX Supplement-100 mL	LIFE Technologies	35050038
HBSS, calcium, magnesium, no phenol red-500 mL	LIFE Technologies	14025092
Ibidi Gas incubation system for CO2 and O2	Ibidi	11922
Ibidi Heating system	Ibidi	10915
Leica SP8 upright microscope – Multiphoton excitation 680–1300 nm	Leica		Equipped with a 25x water-dipping objective (HC IRAPO L 25x/1.00 W) in combination with a tunable laser (Spectra-Physics, InSight DS + Single)
Non Essential Amino Acids	LIFE Technologies	11140035
NuSieve GTG Agarose ,25 g	Biozym /Lonza	859081
OCT Embedding Matrix	Carlroth	6478.1
Paraformaldehyde, 16% W/V AQ. 10 x10 mL	VWR International	43368.9M
Pen-Strep	Gibco	15140122
Stiefel Biopsy-Punch 2 mm	Stiefel	270130
Straight Sharp/Sharp Dissecting Scissors 11.4 cm	Fisher Scientific	15654444
Thimerosal Bioxtra, 97%–101%	Sigma-Aldrich	T8784-1G
Zeiss Axioimager M2 upright microscope	Zeiss