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Correspondence: Adelheid Elbe-Bürger, Department of Dermatology, University Hospital Vienna, Medical University of Vienna, Waehringer Guertel 18-20, Vienna 1090, Austria.
Human skin consists of three compartments, each endowed with a particular structure and the presence of several immune and nonimmune cells that together comprise a protective shield and orchestrate multiple processes in the skin. Appropriate processing of human skin samples acquired from healthy volunteers or patients is essential for successful analysis in basic, translational, and clinical research to obtain accurate and reliable results, despite differences between individuals. From the wide range of available assays and methods, it is necessary to select the suitable method for separation of skin compartments, which will provide preservation or high viability of skin cells or whole structures that will be analyzed or further processed. In this paper, we review and discuss skin separation methods and compare their features such as processing time, cell viability, location of the basement membrane after detachment of the epidermis from the dermis, and their application. Furthermore, we visualize different cell populations and structures in epidermal and dermal sheets using confocal microscopy. It is aimed to provide an overview of the optimal processing of human skin samples and their possible application.
Skin is a complex and dynamic organ acting as an efficient protective barrier from the external environment. It can be divided into three structural compartments (epidermis, dermis, and hypodermis) (Figure 1a) on the basis of various communicating cell populations of predominantly keratinocytes (KCs) and fibroblasts as well as melanocytes, Merkel cells, nerve cells, adipocytes, endothelial cells, and diverse immune cells. Regardless of the substantial amount of knowledge about human skin and its components, there are still many aspects being currently investigated and will be in the future. Human skin samples are widely used for research and diagnostic purposes to study the occurrence, location, phenotype, and function of immune and nonimmune cell types or structures such as nerves, lymphatic/blood vessels, and appendages as well as for pharmacology and toxicology studies. The use of epidermal or dermal sheets of the desired size can be beneficial and important for research and clinical studies to study cells and structures in their environment and in the development of diagnostic tools and research techniques in dermatology.
Summary Points
Advantages
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Relatively low-key ethical considerations using ex vivo skin because it is obtained usually as surgical waste from reductive surgeries.
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Simplicity and efficacy of this model (skin sheets) and particular skin separation methods in research and clinic.
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Small sample size (punch biopsy/piece of skin) is sufficient to stain and image skin compartments of healthy or diseased individuals.
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Improvement of the diagnostic accuracy (e.g., ex vivo skin sheets allow studying standardized re-epithelialization during wound healing).
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Applicable in the clinic for minimally invasive and scarless skin sampling in patients.
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Dynamics of infections caused by pathogens can be studied, immune responses can be quantified, and antimicrobial agents can be tested.
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Further sample processing and isolation of desired cell populations for analysis or cultivation are feasible.
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Imaging of skin sheets enables studying the morphology, phenotype, occurrence, and enumeration of cell populations on a larger surface than for instance in skin sections.
Limitations
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Selection of an unsuitable skin separation method for a desired analysis can either damage cells and skin structures or lead to disintegration of skin sheets.
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No systemic circulation and recruitment of peripheral cells, liquids, and factors are possible in skin sheet explants.
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Owing to the thickness of skin sheets, the employment of confocal microscopy or different imaging techniques (with high resolution and the potential to penetrate the tissue) might be necessary and are not always available.
Figure 1Selection of human skin separation methods and the location of the BM on skin sections and sheets.(a) An overview of skin sample excision and skin compartment separation methods. (b) Location of the BM after enzymatic, chemical, and mechanical detachment of the E from the D. Representative immunofluorescent images depict the location of collagen IV (magenta) in skin sections as well as in sheets. Bar = 10 μm. D, dermis; BM, basement membrane; E, epidermis; h, hour; H, hypodermis; min, minute; NH4SCN, ammonium thiocyanate; ON, overnight; RT, room temperature.
Skin obtained from healthy volunteers or patients can be used for the separation of the epidermis from the dermis and their analysis depending on factors and parameters needed (Figure 1a, panel 1). Skin can be sampled as (i) stripe (scissor, scalpel), (ii) shave biopsy (dermatome), or (iii) punch biopsy of different sizes and thicknesses (biopsy punch) (Figure 1a, panel 2). Owing to the comprehensive methodology and processing repertoire for skin studies, it is important to select the appropriate method for separation of skin compartments and to consider selected parameters such as the viability of skin cells and preservation of markers (Table 1). Depending on the separation method, epidermal and dermal sheets can be further used for imaging using distinct techniques: ex vivo culture; in vitro explant culture; or isolation of cell populations of interest for diverse applications such as transplantation, flow cytometry analysis, or multiple omics (e.g., genomics, transcriptomics, proteomics, metabolomics, lipidomics, and many others) (Table 1 and Supplementary Table S1). Employment of skin sheets enables the visualization of a transverse skin plane and a clear analysis of cell populations in their environment because it allows not only the identification of their exact location and morphology but also provides a larger surface for the quantification (e.g., enumeration) of skin cells. Of note, the separate processing of skin sheets allows a better enrichment of scarce cell types in the respective compartments. Furthermore, it decreases the risk of misidentification of cell populations when analyzing single-cell suspensions of separated compartments. Dissociation of whole skin using commercially available predefined enzymes (e.g., Miltenyi products) without previous separation of skin compartments are trendy and efficient, yet not suitable for all cell types (
). In contrast to skin sheets, the analysis of the frontal plane of skin cross-sections (of diverse thicknesses) enables mostly partial visualization and capture of cell populations and structures in the skin. At this point, it is mandatory to mention that the adult human has three types of hair: terminal, vellus, and intermediate. Terminal hairs are thick and pigmented (scalp, beard/eyebrows, axilla, pubic); vellus hairs are thin, short, and unpigmented (cover the whole body, except for the glabrous skin); and intermediate hairs are a combination of terminal and vellus hairs (e.g., arms and legs) (
). Accordingly, processing of hairy skin areas might require hair removal, by either plucking or shaving before epidermal/dermal separation. The most popular hair-extraction technique is plucking vellus or terminal hair from full-thickness skin (
). Of note, for studies involving the analysis of intact hair or hair follicles (HFs), it is important to consider that plucking hair (especially in the anagen phase) usually leaves the dermal sheath and papillae of the HFs in the dermis or the hypodermis (
). To enable dermal papillae extraction from the tissue, incubation with an enzyme such as dispase (Figure 1a) might be required beforehand to loosen the collagenous matrix of the outer dermal sheath (
) for either hair grafting, transplantation, or research purpose. The follicle unit comprises 1‒4 HFs with sebaceous glands that can be obtained through direct excision using punch biopsies (Figure 1a) or stereomicroscope dissection (
Table 1Benefits and Limitations of Selected Epidermal/Dermal Separation Methods
Separation Method
Agent or Treatment
Benefits
Limitations
Enzymatic Basal membrane remains mainly on the dermis
Dispase
High yields of viable skin cells Intact skin sheets Skin separation under physiologic conditions Flexible incubation time Applicable for explant culture and skin grafting Applicable for single-cell analysis, omics technology, or culture (epidermis and dermis) Low medium costs
Potential impairment of certain cell membrane proteins (e.g., surface markers) Excessive time or concentration can damage tissue
Trypsin
Moderate cell viability Short incubation time Useful for single-cell analysis or single-cell culture (epidermis and dermis) Moderate medium costs
Disintegrated epidermal sheet Harm of certain cell membrane proteins (e.g., pan marker epitopes, adhesion molecules) Excessive time or concentration can damage tissue
Chemical (neutral salts) Vast majority of the basal membrane remains on the dermis
NH4SCN
Tissue and cell fixation Intact skin sheets Enables removal of the epidermis in fixed skin sections (e.g., after ex vivo culture) Preparation of high-quality (i.e., intact) RNA and DNA or for imaging purposes Short incubation time Low cost
Not appropriate for explantation and cell cultivation
NaCl
Tissue and cell preservation Intact skin sheets Preparation of high-quality (i.e., intact) RNA and DNA or for imaging purposes Self-antigen preservation (e.g., bullous pemphigoid) Simplicity and efficacy Low cost
Not applicable for explant culture and cell cultivation Low cell viability Interference with cellular electrolytic equilibrium Longer incubation time is required than for enzymatic treatment
EDTA
Intact skin sheets In combination with trypsin, single-cell suspensions can be prepared from epidermal sheets for imaging purposes Low cost
Not applicable for explant culture and cell cultivation Longer incubation time may be required than for enzymatic treatment
Mechanical Basal membrane is split between the epidermis and dermis
Suction
High viability of skin cells Lack of chemical violation of skin cells Standardized blister size Minimal invasive and scarless skin sampling in vivo compared with biopsy punches Epidermis is applicable for skin grafting Preparation of high-quality (i.e., intact) RNA and DNA or for imaging purposes Can be utilized as wound model ex vivo Low cost
Long incubation time ex vivo Suction of dermal immune cells into the epidermis
Heat
Low viability of skin cells Applicable as wound model ex vivo Simplicity and efficacy Short incubation time Low cost
High incubation temperature
Stretching
Lack of chemical violation of skin cells Low cost
Induction of mechanical stress
Milling cutter
Viable and intact dermal sheet Lack of chemical violation of dermal cells Applicable as wound and infection model ex vivo Short incubation time
Epidermal‒dermal separation and technical considerations
Different separation strategies of skin compartments ex vivo aim to detach the epidermis from the dermis in a meticulous manner. The epidermis is attached to the dermis through the basement membrane (BM), which is a mesh-like structure that connects basal epidermal KCs through extracellular matrix proteins such as laminins and collagens (resembling lamina lucida and lamina densa), with the sublamina densa region representing the upper compartment of the papillary dermis (
). There are three major categories for the dissociation of the epidermal‒dermal junction: (i) enzymatic, (ii) chemical, and (iii) mechanical (Figure 1a, panel 3) (reviewed by
The enzymatic separation is based on the application of proteolytic enzymes such as dispase that during indicated incubation conditions (Figure 1a, panel 3) cleaves the collagen proteins within the BM, leading to smooth separation of the epidermal and dermal compartments with high yields of viable cell populations and traces of the BM on epidermal sheets, which are barely detectable in skin sections (Figure 1b, left column). Employment of enzymes for epidermal/dermal separation might result in lodging the HF in the dermis (enables the studying of preserved and intact structures such as sebaceous glands and sweat glands), leaving the epidermis hair free (epidermal sheets from areas with high-hair density might be perforated).
For chemical separation, neutral salts such as ammonium thiocyanate (NH4SCN) and sodium chloride or acids such as EDTA are employed (Figure 1a, panel 3), dissolving proteins at basal KC cell membranes above the BM (Figure 2b, three middle columns) (
) or even cell death through cell fixation. Thus, chemical separation methods are not recommended for further cultivation of the separated skin compartments or cells but are applicable for the preparation of high-quality (i.e., intact) RNA and DNA or for imaging purposes (e.g., NH4SCN) (
Figure 2Identification and visualization of cell populations and structures in epidermal and dermal sheets. (a, b) The three-dimensional projections of distinct immunofluorescent-labeled (a) epidermal and (b) dermal cell populations are shown in skin sheets.
). Negative pressure is applied on the skin, resulting in blister formation and consequently detachment of the epidermis from the dermis within the BM (upper lamina densa/lamina lucida), leaving, for example, collagen IV proteins on the dermal part and traces on epidermal sheets (
) (Figure 1b, right column). In contrast to in vivo skin sampling using punch biopsy, skin suction is a minimally invasive, nonscarring sampling technique where epidermal roofs can be used for staining or grafting on patients and the suction blister fluid studied with omics technology (
). Because no chemical violation of skin cells ensues, viable cells with preserved markers in skin sheets permit further processing (Supplementary Table S1). Employment of this method on ex vivo skin allows standardized re-epithelialization studies during wound healing. Of note, the BM within the wound bed is not equally lifted (Supplementary Figure S1). The long waiting time for blister formation ex vivo might be a disadvantage in terms of potential degradation of particular molecules/proteins/factors/cytokines over the incubation period at room temperature as well as the suction of dermal immune cells in the area below the forming blister into the epidermal blister roof (
) and needs to be considered depending on the question raised. Furthermore, employment of mechanical epidermal/dermal separation methods such as skin blistering is better suitable for skin regions with low hair density (distal parts of the body such as the forearm, inner thighs, and back). The heating technique is quite fierce and invasive, where skin samples are warmed up to 50‒60 °C in a water bath or through the application of an external heat source (Figure 1a, panel 3). This results in blister formation and therefore a dissociation of the epidermis from the dermis within the BM through likely the destabilization of collagen fibers in the lamina densa or below (
). The advantage of this technique is the short incubation time and an opportunity to study the healing processes of burn wounds of low-grade ex vivo with possible expression and secretion of heat-shock proteins in response to high temperature and tissue remodeling after blistering. However, incubation of skin at a temperature higher than that at physiologic conditions (37‒39 °C) can induce cell death, secretion of heat-shock proteins resembling stress conditions, and degradation of certain protein structures and DNA damage (
). Another possibility to obtain unperturbed, dermal sheets with viable cells is to use a rotating ball-shaped milling cutter ex vivo. This method allows the removal of the epidermis from the dermis within seconds and can be used as a valuable tool to examine the mechanisms of host‒pathogen interactions combined with the option to test antimicrobial agents directly in human tissue (
) is based on stretching thin skin stripes to the limit, fastening them and scratching the surface with a scalpel, and removing the epidermis with forceps. This procedure causes the rupture of the BM and a definite loss of epidermal attachment to the dermis. Because this mechanical approach is quite harsh for skin physiology, this technique is rarely used. Skin separation protocols and the protocol for Figure 1 can be found in Supplementary Text S1 and S2, respectively. The benefits and limitations of selected epidermal/dermal separation methods discussed earlier are summarized in Table 1.
Identification of cell populations in epidermal and dermal sheets
Visualization of cell populations in skin sheets as well as dermal structures such as blood and lymph vessels, appendages, and nerves can be achieved by marking molecules expressed by cell or structure of interest (the so-called pan markers) with small molecules or antibodies (e.g., fluorescently labeled) that can be further detected under a fluorescence microscope (Figure 2). Imaging of epidermal sheets enables studying cells such as resident Langerhans cells (LCs) with their long intercellular dendrites (Figure 2a); an efficient and appropriate quantification of their number (
Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.
). Imaging of epidermal sheets, representing a mighty area in comparison with that of skin sections, enables the identification of rarely occurring cell populations such as resident T cells, melanocytes, Merkel cells (Figure 2a), or proliferative KCs in the healthy or diseased epidermis in steady state or after previous treatment and cultivation with pattern recognition receptor agonists (
Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.
). To investigate dermal cell populations, spatial imaging is suggested owing to the thickness of samples. Three-dimensional imaging facilitates an accurate visualization of Schwann cells (Figure 2b), terminal axons (
), and blood and lymphatic vessels along with immune cell populations such as T cells, dendritic cells (DCs), macrophages, and mast cells (MCs) in the dermis and nonimmune cells such as fibroblasts (
Another possibility to analyze immune cells is to cultivate epidermal/dermal sheets and full-thickness or dermatomed skin fragments/punch biopsies for a few days. Resident cells with migratory potential such as LCs (from full-thickness/dermatomed skin biopsies or epidermal sheets) (
Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.
Mast cell migration from the skin to the draining lymph nodes upon ultraviolet irradiation represents a key step in the induction of immune suppression.
) can then be collected from culture wells for further evaluation. They can be analyzed using flow cytometry, imaging flow cytometry (ImageStream), imaging (conventional or confocal microscopy, imaging mass cytometry [cytometry by time of flight tissue imaging]), or omics technology or further cultivated and expanded. Nonmigratory cells such as dermal macrophages can only be analyzed in the tissue. Supplementary Table S1 provides the respective literature, including the description of the epidermal/dermal separation methods and their application in research as well as in clinics.
The employment of modern techniques and software for spatial and quantitative analysis of human skin such as the fluorescence-based multiplex imaging system and automatized TissueFAXS or other systems offer not only spatial cell mapping and quantification but also standardized analysis procedures and consequently previously unreported insights into human skin biology (
Owing to space limitations, unfortunately, we were not able to include and acknowledge all the work of researchers who contributed to this field. We thank Michael Mildner and his team for providing antibodies for the characterization of nerve cells and Merkel cells in human skin used in this study. This work was supported by the Austrian Science Fund (P31485-B30 and DK W1248-B30 both to AEB).
Supplementary Materials and Methods. Supplementary Text S1. Skin Separation Protocols
Equipment, tools, and reagents used for human skin sampling, excision, and processing
For skin sampling and excision, the equipment and tools used are shown in Supplementary Table S2, and the reagents commonly used for disinfection of the skin surface are shown in Supplementary Table S3.
Examples of procedures used for the separation of skin compartments
Enzymatic skin separation
For dispase II ex vivo procedure, human skin with an adipose layer is transported on 4 °C cooling packs to the laboratory and processed within 1‒6 hours after abdominal or breast surgery. For compartment separation, the enzyme dispase II (Roche Diagnostics, Basel, Switzerland) should be reconstituted according to the manufacturer’s instructions and thereafter diluted in RPMI or DMEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA) to a concentration of 1.2‒1.8 U/ml. It is recommended to add antibiotics (e.g., 1% penicillin‒streptavidin, Gibco, Thermo Fisher Scientific) to the cell culture media, whereas no fetal bovine serum/fetal calf serum supplementation is required for this step. Skin samples (full-thickness or dermatomed biopsy punches, skin stripes, skin pieces) are floated on the cell culture medium with dispase II or submerged in the solution for 1‒2 hours at 37 °C or overnight at 4 °C. Subsequently, skin samples can be washed with Dulbecco’s PBS (DPBS) (Thermo Fisher Scientific), and then the epidermis is separated from the dermis using two precision tweezers with sharp tips (one to hold the dermis and another one for pulling down the epidermis). Epidermal and dermal sheets can be (i) fixed, stained, and analyzed; (ii) cultivated and then processed according to the need; and (iii) incubated with enzymes to obtain single-cell suspensions for either single-cell cultivation or immediate analysis (flow cytometry, cytometry by time of flight, adhesion slides, single-cell sequencing, and others).
For sample fixation, epidermal and dermal sheets can be fixed with 4‒4.5% formaldehyde (FA; Liquid Production GmbH, Flintsbach an Inn, Germany) (2 hours, 4 °C). FA-fixed samples need to be additionally permeabilized with 0.5% Triton X-100 in Tris-buffered saline (TBS) (45 minutes, 4 °C). Alternatively, epidermal sheets can be fixed with ice-cold acetone (Carl Roth) (10‒20 minutes at room temperature [RT]) and further processed or stored at ‒80 °C for later analysis.
The reagents and tools applied for the separation and fixation of skin compartments using dispase are shown in Supplementary Table S4.
Chemical skin separation
For ammonium thiocyanate (NH4SCN; Carl Roth GmbH + Co. KG, Germany) procedure, skin samples are placed on a 3.8% NH4SCN in DPBS solution and incubated at 37 °C for 1 hour. Subsequently, the skin can be washed in DPBS, and then the epidermis can be separated from the dermis using two precision tweezers with sharp tips as described in the section on dispase II ex vivo procedure and further processed. For sample fixation, epidermal and dermal sheets are already fixed but can additionally be fixed with 4-4.5% FA or acetone (see the section on dispase II ex vivo procedure). The reagents and tools applied for the separation of skin compartments using NH4SCN are shown in Supplementary Table S5.
For sodium chloride (NaCl; MERCK) procedure, skin samples are floated on or submerged in a 1 M NaCl solution for 24 hours at RT (∼22 ºC) or 48 hours at 4 °C. Thereafter, the skin can be washed in DPBS or double-distilled water (ddH2O), and then the epidermis can be separated from the dermis as described in the section on dispase II ex vivo procedure and further processed. For sample fixation, epidermal and dermal sheets are already fixed but can additionally be fixed with 4‒4.5% FA (2 hours, 4 °C) and permeabilized with 0.5% Triton X-100 in TBS (45 minutes, 4 °C). The reagents and tools applied for the separation of skin compartments using NaCl are shown in Supplementary Table S6.
For EDTA (Invitrogen, Thermo Fisher Scientific) procedure, skin samples according to the needs are floated on or submerged in the 2 mM EDTA solution for 24 hours at RT (∼22 ºC) or 48 hours at 4 °C. Thereafter, the skin can be washed in DPBS or ddH2O, and the epidermis can be separated from the dermis and processed (as described in the section on dispase II ex vivo procedure). For sample fixation, epidermal and dermal sheets need to be additionally fixed with 4‒4.5% FA (2 hours, 4 °C) and permeabilized with 0.5% Triton X-100 in TBS (45 minutes, 4 °C). The reagents and tools applied for the separation of skin compartments using EDTA are shown in Supplementary Table S7.
Mechanical skin separation
For suction blister in vivo procedure, to produce blisters, a pressure instrument (Electronic Diversities, Finksburg, MD) applying pressure (150‒200 mmHg) through two sterile, 1‒5-hole (5 mm diameter per hole) skin suction plates, according to the need of the experiment, is mounted airtight onto the inner forearm of healthy volunteers. Development of the suction blisters usually requires between 2 and 3 hours, depending on the individual. The blister roof (=epidermis) can be removed using scissors, and the blister fluid (typically between 20 and 30 μl in volume per blister) can be collected with a Micro-Fine syringe (30 G needle) from visually intact and blood-uncontaminated blisters in an Eppendorf tube containing a protease inhibitor mixture and then both used for further analysis (
For suction blister ex vivo procedure, human skin from plastic surgeries (∼10 × 10 cm) should be disinfected with an antiseptic agent (e.g., kodan forte farblos, Schülke & Mayr, Norderstedt, Germany) and wiped with sterile gauze. Next, the subcutaneous fat has to be removed using scissors, and the skin is placed on a styrofoam pad wrapped with aluminum foil (skin fixation base). One sterile orifice plate with five holes (5 mm diameter per hole) needs to be attached in the middle of the skin piece, and the pressure instrument (Electronic Diversities) is set to 200‒250 mmHg for 6‒8 hours. The blister roof and the blister liquid can be harvested as described (see the section on suction blister in vivo procedure) and used for further analysis (culture and/or staining, omics technology). The dermis can be used for ex vivo re-epithelialization, infection studies, and others (
). For staining purposes, epidermal and dermal sheets need to be additionally fixed with 4‒4.5% FA (2 hours, 4 °C) and permeabilized with 0.5% Triton X-100 in TBS (45 minutes, 4 °C) or alternatively, fixed with ice-cold acetone (10‒20 minutes, RT) according to the need of the experiment. The equipment, tools, and reagents applied for the separation of skin compartments using low pressure for skin blister initiation are shown in Supplementary Table S8.
For milling cutter ex vivo procedure, human skin tissue from surgeries should be disinfected and cut with scissors to the required size. The adipose layer can be totally or partially removed with scissors, and the remaining skin sample can be mounted onto a styrofoam pad wrapped with aluminum foil using syringe needles. Application of a rotating ball-shaped milling cutter (6 mm, Proxxon, Wecker, Luxemburg) fixed on a dental micro motor handpiece (Marathon N7, TPC Advanced Technology, Diamond Bar, CA) at 16,000 r.p.m. removes the epidermis with an area of ∼5 × 5 mm within seconds, thus creating a superficial wound. Dermal sheets can be further cultivated for re-epithelialization or infection studies and/or fixed with 4‒4.5% FA (for 2 hours, 4 °C) and permeabilized with 0.5% Triton X-100 in TBS (45 minutes, 4 °C) for staining purposes (
). The devices and tools for the separation of skin compartments using a milling cutter are shown in Supplementary Table S9.
For heat procedure, human skin samples are placed into preheated (60 °C) DPBS for 45‒60 seconds and then transferred immediately into cooled DPBS. Subsequently, the epidermis can be separated from the dermis using two precision tweezers (as described in the section on dispase II ex vivo procedure) for further processing (
). For sample fixation, epidermal and dermal sheets can be fixed with 4‒4.5% FA (2 hours, 4 °C) and additionally permeabilized with 0.5% Triton X-100 in TBS (45 minutes, 4 °C). Alternatively, epidermal sheets can be fixed with ice-cold acetone (10‒20 minutes, RT) and further processed or stored at ‒80 °C for future analysis. The devices and reagents applied for the separation of skin compartments using heat are shown in Supplementary Table S10.
Supplementary Text S2. Protocols for Figures 1 and 2
Skin sample excision, processing, and analysis
Skin samples from anonymous healthy female donors (aged 33 and 37 years) were obtained from abdominal plastic surgery. The study was approved by the local ethics committee of the Medical University of Vienna (Vienna, Austria) and conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from both participants.
Experiments were performed within 1‒2 hours after surgery on skin pieces free of injuries, stretch marks, or redness. The protocols for the separation of skin compartments and fixation were used as described in Supplementary Text S1.
Acquisition of images
For imaging of epidermal and dermal sheets, a confocal laser scanning microscope (FLUOVIEW-FV 3000, Olympus, Tokyo, Japan, equipped with OBIS lasers: 405, 488, 561, 640 nm), and Olympus FV31S-SW software was used in this study. Images were acquired with ×10 and ×20 objectives (UPlanXApo) and processed using ImageJ Fiji software (ImageJ, National Institutes of Health, Bethesda, MD) or ImarisViewer (version 9.8; Oxford Instruments) for the three-dimensional projections of epidermal and dermal sheets. Skin sheets were stained using antibodies listed in Supplementary Table S11. Staining procedures were performed essentially as described previously (
). The embedding of skin sheets depends on the scientific question raised. For the purpose of this manuscript, we imaged the transverse and horizontal planes of the epidermis from the stratum basale. The epidermal sheet was placed on a glass slide, with the stratum basale facing the glass slide, coated with an imaging mount (ProLong Gold antifade reagent; Invitrogen, Thermo Fisher Scientific), covered with a cover slip, and imaged as Z-stack using a confocal microscope as described earlier. For visualization of the dermis, a similar procedure was employed. Dermal sheets were imaged from the stratum basale as Z-stack. The dermal sheet was placed in imaging mount, with the stratum basale facing the glass slide and covered with a cover slip. The antibodies and reagents used in this study are shown in Supplementary Table S11.
Supplementary Figure S1Location of the basement membrane within a suction blister wound bed ex vivo. Graphics illustrate the distribution of the basement membrane (collagen IV) within the wound bed, wound bed edge, and wound edge, together with representative immunofluorescent images. Bar = 10 μm. BM, basement membrane; D, dermis; E, epidermis.
Supplementary Figure S2Visualization of selected cell markers on epidermal and dermal cell populations and structures through immunofluorescence staining. Merged and single-channel images of cell markers in (a) epidermal and (b) dermal sheets are shown. α-SMA, α-smooth muscle actin.
Mast cell migration from the skin to the draining lymph nodes upon ultraviolet irradiation represents a key step in the induction of immune suppression.
Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.
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