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Epidermal/Dermal Separation Techniques and Analysis of Cell Populations in Human Skin Sheets

      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.

      Abbreviations:

      BM (basement membrane), DC (dendritic cell), HF (hair follicle), KC (keratinocyte), LC (Langerhans cell), MC (mast cell), NH4SCN (ammonium thiocyanate)

      Introduction

      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

      • Relatively low-key ethical considerations using ex vivo skin because it is obtained usually as surgical waste from reductive surgeries.
      • Simplicity and efficacy of this model (skin sheets) and particular skin separation methods in research and clinic.
      • Small sample size (punch biopsy/piece of skin) is sufficient to stain and image skin compartments of healthy or diseased individuals.
      • Improvement of the diagnostic accuracy (e.g., ex vivo skin sheets allow studying standardized re-epithelialization during wound healing).
      • Applicable in the clinic for minimally invasive and scarless skin sampling in patients.
      • Dynamics of infections caused by pathogens can be studied, immune responses can be quantified, and antimicrobial agents can be tested.
      • Further sample processing and isolation of desired cell populations for analysis or cultivation are feasible.
      • 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

      • 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.
      • No systemic circulation and recruitment of peripheral cells, liquids, and factors are possible in skin sheet explants.
      • 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 thumbnail gr1
      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.

      Available methods

      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 (
      • Philippeos C.
      • Telerman S.B.
      • Oulès B.
      • Pisco A.O.
      • Shaw T.J.
      • Elgueta R.
      • et al.
      Spatial and single-cell transcriptional profiling identifies functionally distinct human dermal fibroblast subpopulations.
      ;
      • Theocharidis G.
      • Tekkela S.
      • Veves A.
      • McGrath J.A.
      • Onoufriadis A.
      Single-cell transcriptomics in human skin research: available technologies, technical considerations and disease applications.
      ;
      • Wang S.
      • Drummond M.L.
      • Guerrero-Juarez C.F.
      • Tarapore E.
      • MacLean A.L.
      • Stabell A.R.
      • et al.
      Single cell transcriptomics of human epidermis identifies basal stem cell transition states.
      ). 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) (
      • Buffoli B.
      • Rinaldi F.
      • Labanca M.
      • Sorbellini E.
      • Trink A.
      • Guanziroli E.
      • et al.
      The human hair: from anatomy to physiology.
      ). 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 (
      • Camidge D.R.
      • Randall K.R.
      • Foster J.R.
      • Sadler C.J.
      • Wright J.A.
      • Soames A.R.
      • et al.
      Plucked human hair as a tissue in which to assess pharmacodynamic end points during drug development studies.
      ;
      • Wagner T.
      • Gschwandtner M.
      • Strajeriu A.
      • Elbe-Bürger A.
      • Grillari J.
      • Grillari-Voglauer R.
      • et al.
      Establishment of keratinocyte cell lines from human hair follicles.
      ). 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 (
      • Chiu H.C.
      • Chang C.H.
      • Wu Y.C.
      An efficient method for isolation of hair papillae and follicle epithelium from human scalp specimens.
      ;
      • Martino P.A.
      • Heitman N.
      • Rendl M.
      The dermal sheath: an emerging component of the hair follicle stem cell niche.
      ). 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 (
      • Guo Z.
      • Draheim K.
      • Lyle S.
      Isolation and culture of adult epithelial stem cells from human skin.
      ;
      • Limbu S.
      • Higgins C.A.
      Isolating dermal papilla cells from human hair follicles using microdissection and enzyme digestion.
      ). Another possibility is the dissection of skin stripes with HFs and their individual processing (
      • Pathomvanich D.
      Donor harvesting: a new approach to minimize transection of hair follicles.
      ) or as HF units (
      • Jimenez F.
      • Alam M.
      • Hernandez I.
      • Poblet E.
      • Hardman J.A.
      • Paus R.
      An efficient method for eccrine gland isolation from human scalp.
      ) 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 (
      • Jiménez F.
      Method for human eccrine sweat gland isolation from the scalp by means of the micropunch technique and vital dyes.
      ;
      • Jimenez F.
      • Alam M.
      • Hernandez I.
      • Poblet E.
      • Hardman J.A.
      • Paus R.
      An efficient method for eccrine gland isolation from human scalp.
      ).
      Table 1Benefits and Limitations of Selected Epidermal/Dermal Separation Methods
      Separation MethodAgent or TreatmentBenefitsLimitations
      Enzymatic

      Basal membrane remains mainly on the dermis
      DispaseHigh 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
      TrypsinModerate 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
      NH4SCNTissue 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
      NaClTissue 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
      EDTAIntact 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
      SuctionHigh 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
      HeatLow viability of skin cells

      Applicable as wound model ex vivo

      Simplicity and efficacy

      Short incubation time

      Low cost
      High incubation temperature
      StretchingLack of chemical violation of skin cells

      Low cost
      Induction of mechanical stress
      Milling cutterViable and intact dermal sheet

      Lack of chemical violation of dermal cells

      Applicable as wound and infection model ex vivo

      Short incubation time
      Lack of epidermis
      Abbreviations: NaCl, sodium chloride; NH4SCN, ammonium thiocyanate.

      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 (
      • Bolognia J.L.
      • Schaffer J.V.
      • Cerroni L.
      Dermatology: 2-Volume Set. 4th ed.
      ). 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
      • Zou Y.
      • Maibach H.I.
      Dermal–epidermal separation methods: research implications.
      ).
      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) (
      • Zou Y.
      • Maibach H.I.
      Dermal–epidermal separation methods: research implications.
      ). Of note, these agents can cause ionic disequilibrium in the tissue and therefore lead to swelling or shrinkage of cells (
      • Baker L.B.
      Physiology of sweat gland function: the roles of sweating and sweat composition in human health.
      ;
      • Woodley D.
      • Sauder D.
      • Talley M.J.
      • Silver M.
      • Grotendorst G.
      • Qwarnstrom E.
      Localization of basement membrane components after dermal-epidermal junction separation.
      ) 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) (
      • Longley J.
      • Ding T.G.
      • Cuono C.
      • Durden F.
      • Crooks C.
      • Hufeisen S.
      • et al.
      Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells.
      ;
      • Tschachler E.
      • Reinisch C.M.
      • Mayer C.
      • Paiha K.
      • Lassmann H.
      • Weninger W.
      Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.
      ).
      Figure thumbnail gr2
      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.
      The mechanical dissociation of skin compartments comprises the following techniques: in vivo (
      • Rojahn T.B.
      • Vorstandlechner V.
      • Krausgruber T.
      • Bauer W.M.
      • Alkon N.
      • Bangert C.
      • et al.
      Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic dermatitis.
      ) and ex vivo (
      • Rakita A.
      • Nikolić N.
      • Mildner M.
      • Matiasek J.
      • Elbe-Bürger A.
      Re-epithelialization and immune cell behaviour in an ex vivo human skin model.
      ) skin suction, heat (
      • Jian L.
      • Cao Y.
      • Zou Y.
      Dermal-epidermal separation by heat.
      ), milling cutter treatment (
      • Schaudinn C.
      • Dittmann C.
      • Jurisch J.
      • Laue M.
      • Günday-Türeli N.
      • Blume-Peytavi U.
      • et al.
      Development, standardization and testing of a bacterial wound infection model based on ex vivo human skin.
      ), and stretching (
      • Van Scott E.J.
      Mechanical separation of the epidermis from the corium.
      ) (Figure 1a, panel 3, not all indicated). The suction method can be used in clinical research (
      • Rojahn T.B.
      • Vorstandlechner V.
      • Krausgruber T.
      • Bauer W.M.
      • Alkon N.
      • Bangert C.
      • et al.
      Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic dermatitis.
      ), translational studies (
      • Polak D.
      • Hafner C.
      • Briza P.
      • Kitzmüller C.
      • Elbe-Bürger A.
      • Samadi N.
      • et al.
      A novel role for neutrophils in IgE-mediated allergy: evidence for antigen presentation in late-phase reactions.
      ), and basic research (
      • Rakita A.
      • Nikolić N.
      • Mildner M.
      • Matiasek J.
      • Elbe-Bürger A.
      Re-epithelialization and immune cell behaviour in an ex vivo human skin model.
      ). 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 (
      • Manabe M.
      • Naito K.
      • Ikeda S.
      • Takamori K.
      • Ogawa H.
      Production of blister in normal human skin in vitro by blister fluids from epidermolysis bullosa.
      ;
      • Saksela O.
      • Alitalo K.
      • Kiistala U.
      • Vaheri A.
      Basal lamina components in experimentally induced skin blisters.
      ) (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 (
      • Rojahn T.B.
      • Vorstandlechner V.
      • Krausgruber T.
      • Bauer W.M.
      • Alkon N.
      • Bangert C.
      • et al.
      Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic dermatitis.
      ) or in clinics to treat vitiligo (
      • Gao P.R.
      • Wang C.H.
      • Lin Y.J.
      • Huang Y.H.
      • Chang Y.C.
      • Chung W.H.
      • et al.
      A comparative study of suction blister epidermal grafting and automated blister epidermal micrograft in stable vitiligo.
      ). 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 (
      • Rakita A.
      • Nikolić N.
      • Mildner M.
      • Matiasek J.
      • Elbe-Bürger A.
      Re-epithelialization and immune cell behaviour in an ex vivo human skin model.
      ) 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 (
      • Zou Y.
      • Maibach H.I.
      Dermal–epidermal separation methods: research implications.
      ). 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 (
      • Purschke M.
      • Laubach H.J.
      • Anderson R.R.
      • Manstein D.
      Thermal injury causes DNA damage and lethality in unheated surrounding cells: active thermal bystander effect.
      ). 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 (
      • Schaudinn C.
      • Dittmann C.
      • Jurisch J.
      • Laue M.
      • Günday-Türeli N.
      • Blume-Peytavi U.
      • et al.
      Development, standardization and testing of a bacterial wound infection model based on ex vivo human skin.
      ). Finally, stretching of the skin or the so-called stripping off epidermis (
      • Van Scott E.J.
      Mechanical separation of the epidermis from the corium.
      ) 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 (
      • Tajpara P.
      • Schuster C.
      • Schön E.
      • Kienzl P.
      • Vierhapper M.
      • Mildner M.
      • et al.
      Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.
      ), morphology, or phenotype; or colocalization with other cells or molecules (
      • Cichoń M.A.
      • Pfisterer K.
      • Leitner J.
      • Wagner L.
      • Staud C.
      • Steinberger P.
      • et al.
      Interoperability of RTN1A in dendrite dynamics and immune functions in human 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 (
      • Cichoń M.A.
      • Pfisterer K.
      • Leitner J.
      • Wagner L.
      • Staud C.
      • Steinberger P.
      • et al.
      Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells.
      ;
      • Tajpara P.
      • Schuster C.
      • Schön E.
      • Kienzl P.
      • Vierhapper M.
      • Mildner M.
      • et al.
      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 (
      • Tschachler E.
      • Reinisch C.M.
      • Mayer C.
      • Paiha K.
      • Lassmann H.
      • Weninger W.
      Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.
      ), 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 (
      • Wang X.N.
      • McGovern N.
      • Gunawan M.
      • Richardson C.
      • Windebank M.
      • Siah T.W.
      • et al.
      A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels.
      ). Blood and lymphatic vessels can be also characterized in the skin sections of developing as well as adult skin (Figure 2b) (
      • Schuster C.
      • Mildner M.
      • Botta A.
      • Nemec L.
      • Rogojanu R.
      • Beer L.
      • et al.
      Development of blood and lymphatic endothelial cells in embryonic and fetal human skin.
      ); however, the thickness of skin sections is a limitation and results in the acquisition only of a part of vessels.
      The protocol for Figure 2 can be found in Supplementary Text S2. In Supplementary Figure S2, the unmerged images of cell markers in epidermal (Figure S2a) and dermal sheets (Figure S2b) are presented.
      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) (
      • Lenz A.
      • Heine M.
      • Schuler G.
      • Romani N.
      Human and murine dermis contain dendritic cells: isolation by means of a novel method and phenotypical and functional characterization.
      ;
      • Tajpara P.
      • Schuster C.
      • Schön E.
      • Kienzl P.
      • Vierhapper M.
      • Mildner M.
      • et al.
      Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.
      ), naïve/memory T cells (from fetal or adult healthy and diseased skin fragments [
      • Clark R.A.
      • Chong B.F.
      • Mirchandani N.
      • Yamanaka K.
      • Murphy G.F.
      • Dowgiert R.K.
      • et al.
      A novel method for the isolation of skin resident T cells from normal and diseased human skin.
      ;
      • Reitermaier R.
      • Krausgruber T.
      • Fortelny N.
      • Ayub T.
      • Vieyra-Garcia P.A.
      • Kienzl P.
      • et al.
      αβγδ T cells play a vital role in fetal human skin development and immunity.
      ]), dermal DCs (
      • Haniffa M.
      • Gunawan M.
      • Jardine L.
      Human skin dendritic cells in health and disease.
      ), or MCs (
      • Byrne S.N.
      • Limón-Flores A.Y.
      • Ullrich S.E.
      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 (
      • Meshcheryakova A.
      • Mungenast F.
      • Ecker R.
      • Mechtcheriakova D.
      Tissue image cytometry. Imaging modalities for biological and preclinical Research: a compendium, volume 1. Part I: Ex vivo biological imaging.
      ).
      No single method can serve all purposes, and the proper skin separation method has to be selected according to the experimental question.

      ORCIDs

      Małgorzata Anna Cichoń: http://orcid.org/0000-0001-9824-9207

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      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).

      Author Contributions

      Conceptualization: MAC, AEB; Funding Acquisition: AEB; Investigation: MAC; Methodology: MAC; Supervision: AEB; Visualization: MAC; Writing - Original Draft Preparation: MAC; Writing - Review and Editing: 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 (
      • Hoetzenecker W.
      • Ecker R.
      • Kopp T.
      • Stuetz A.
      • Stingl G.
      • Elbe-Bürger A.
      Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis.
      ;
      • Müller A.C.
      • Breitwieser F.P.
      • Fischer H.
      • Schuster C.
      • Brandt O.
      • Colinge J.
      • et al.
      A comparative proteomic study of human skin suction blister fluid from healthy individuals using immunodepletion and iTRAQ labeling.
      ;
      • Polak D.
      • Hafner C.
      • Briza P.
      • Kitzmüller C.
      • Elbe-Bürger A.
      • Samadi N.
      • et al.
      A novel role for neutrophils in IgE-mediated allergy: evidence for antigen presentation in late-phase reactions.
      ;
      • Rojahn T.B.
      • Vorstandlechner V.
      • Krausgruber T.
      • Bauer W.M.
      • Alkon N.
      • Bangert C.
      • et al.
      Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type–specific immune regulation in atopic dermatitis.
      )
      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 (
      • Rakita A.
      • Nikolić N.
      • Mildner M.
      • Matiasek J.
      • Elbe-Bürger A.
      Re-epithelialization and immune cell behaviour in an ex vivo human skin model.
      ). 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 (
      • Schaudinn C.
      • Dittmann C.
      • Jurisch J.
      • Laue M.
      • Günday-Türeli N.
      • Blume-Peytavi U.
      • et al.
      Development, standardization and testing of a bacterial wound infection model based on ex vivo human skin.
      ). 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 (
      • Jian L.
      • Cao Y.
      • Zou Y.
      Dermal-epidermal separation by heat.
      ). 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 (
      • Cichoń M.A.
      • Pfisterer K.
      • Leitner J.
      • Wagner L.
      • Staud C.
      • Steinberger P.
      • et al.
      Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells.
      ;
      • Tschachler E.
      • Reinisch C.M.
      • Mayer C.
      • Paiha K.
      • Lassmann H.
      • Weninger W.
      Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.
      ). 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.
      Figure thumbnail fx1
      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.
      Figure thumbnail fx2
      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.
      Supplementary Table S1Examples of the Characterization and Application of Skin Separation Methods
      Skin Separation MethodsApplication in Basic, Translational, and Clinical Research and DiagnosticsReferences
      Owing to space limitations, we were not able to include and acknowledge all the work of researchers who contributed to this field.
      EnzymaticDispaseThe influence of CD26+ and CD26 fibroblasts on the regeneration of human dermo‒epidermal skin substitutes.
      • Michalak-Micka K.
      • Klar A.S.
      • Dasargyri A.
      • Biedermann T.
      • Reichmann E.
      • Moehrlen U.
      The influence of CD26+ and CD26− fibroblasts on the regeneration of human dermo-epidermal skin substitutes.
      Ex vivo infection of human skin with herpes simplex virus 1 reveals mechanical wounds as insufficient entry portals through the skin surface.
      • De La Cruz N.C.
      • Möckel M.
      • Wirtz L.
      • Sunaoglu K.
      • Malter W.
      • Zinser M.
      • et al.
      Ex vivo infection of human skin with herpes simplex virus 1 reveals mechanical wounds as insufficient entry portals via the skin surface.
      Dermal‒epidermal separation by enzyme.
      • Jian L.
      • Cao Y.
      • Zou Y.
      Dermal-epidermal separation by enzyme.
      Spatially and cell-type resolved quantitative proteomic atlas of healthy human skin.
      • Dyring-Andersen B.
      • Løvendorf M.B.
      • Coscia F.
      • Santos A.
      • Møller L.B.P.
      • Colaço A.R.
      • et al.
      Spatially and cell-type resolved quantitative proteomic atlas of healthy human skin.
      Conducting spatial and single-cell transcriptional profiling with human dermal cell populations with focus on fibroblast subpopulations.
      • Philippeos C.
      • Telerman S.B.
      • Oulès B.
      • Pisco A.O.
      • Shaw T.J.
      • Elgueta R.
      • et al.
      Spatial and single-cell transcriptional profiling identifies functionally distinct human dermal fibroblast subpopulations.
      Treatment of giant congenital melanocytic nevi with enzymatically separated epidermal sheet grafting (in vivo)
      • Kishi K.
      • Ninomiya R.
      • Okabe K.
      • Konno E.
      • Katsube K.
      • Imanishi N.
      • et al.
      Treatment of giant congenital melanocytic nevi with enzymatically separated epidermal sheet grafting.
      Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.
      • Lukas M.
      • Stössel H.
      • Hefel L.
      • Imamura S.
      • Fritsch P.
      • Sepp N.T.
      • et al.
      Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.
      Isolation of high-quality mRNA from fixed epidermal sheets and sorted epidermal cells.
      • Longley J.
      • Ding T.G.
      • Cuono C.
      • Durden F.
      • Crooks C.
      • Hufeisen S.
      • et al.
      Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells.
      TrypsinBasal detachment of the epidermis using dispase: tissue spatial organization and fate of integrin α6β4 and hemidesmosomes.
      • Poumay Y.
      • Roland I.H.
      • Leclercq-Smekens M.
      • Leloup R.
      Basal detachment of the epidermis using dispase: tissue spatial organization and fate of integrin α6β4 and hemidesmosomes.
      Localization of basement membrane components after dermal‒epidermal junction separation.
      • Woodley D.
      • Sauder D.
      • Talley M.J.
      • Silver M.
      • Grotendorst G.
      • Qwarnstrom E.
      Localization of basement membrane components after dermal-epidermal junction separation.
      ChemicalNH4SCNA mechanistic view on the aging human skin through ex vivo layer-by-layer analysis of mechanics and microstructure of facial and mammary dermis.
      • Lynch B.
      • Pageon H.
      • Le Blay H.
      • Brizion S.
      • Bastien P.
      • Bornschlögl T.
      • et al.
      A mechanistic view on the aging human skin through ex vivo layer-by-layer analysis of mechanics and microstructure of facial and mammary dermis.
      Rapid, high-quality, and epidermal-specific isolation of RNA from human skin.
      • Trost A.
      • Bauer J.W.
      • Lanschützer C.
      • Laimer M.
      • Emberger M.
      • Hintner H.
      • et al.
      Rapid, high-quality and epidermal-specific isolation of RNA from human skin.
      Characterization of the nerves in human skin sheets.
      • Tschachler E.
      • Reinisch C.M.
      • Mayer C.
      • Paiha K.
      • Lassmann H.
      • Weninger W.
      Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.
      NaClDermal‒epidermal separation by chemical.
      • Jian L.
      • Cao Y.
      • Zou Y.
      Dermal-epidermal separation by chemical.
      Rapid, high-quality, and epidermal-specific isolation of RNA from human skin.
      • Trost A.
      • Bauer J.W.
      • Lanschützer C.
      • Laimer M.
      • Emberger M.
      • Hintner H.
      • et al.
      Rapid, high-quality and epidermal-specific isolation of RNA from human skin.
      Comparative study of autoantigens for various bullous skin diseases by immunoblotting using different dermo‒epidermal separation techniques.
      • Ohata Y.
      • Hashimoto T.
      • Nishikawa T.
      Comparative study of autoantigens for various bullous skin diseases by immunoblotting using different dermo-epidermal separation techniques.
      An ultrastructural comparison of dermo‒epidermal separation techniques.
      • Willsteed E.M.
      • Bhogal B.S.
      • Das A.
      • Bekir S.S.
      • Wojnarowska F.
      • Black M.M.
      • et al.
      An ultrastructural comparison of dermo-epidermal separation techniques.
      MechanicalSuction blisters: in vivoSingle-cell sequencing analysis of the skin samples of healthy individuals and patients with atopic dermatitis using suction blistering.
      • Rojahn T.B.
      • Vorstandlechner V.
      • Krausgruber T.
      • Bauer W.M.
      • Alkon N.
      • Bangert C.
      • et al.
      Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type–specific immune regulation in atopic dermatitis.
      Application of suction blistering for the analysis of immune cell types in the skin of healthy individuals and patients with allergy.
      • Polak D.
      • Hafner C.
      • Briza P.
      • Kitzmüller C.
      • Elbe-Bürger A.
      • Samadi N.
      • et al.
      A novel role for neutrophils in IgE-mediated allergy: evidence for antigen presentation in late-phase reactions.
      Suction blister epidermal graft (SBEG) - an easy way to apply this method.
      • Angeletti F.
      • Kaufmann R.
      Suction blister epidermal graft (SBEG) – an easy way to apply this method..
      Epidermal grafting for wound healing: a review on the harvesting systems, the ultrastructure of the graft, and the mechanism of wound healing.
      • Kanapathy M.
      • Hachach-Haram N.
      • Bystrzonowski N.
      • Connelly J.T.
      • O’Toole E.A.
      • Becker D.L.
      • et al.
      Epidermal grafting for wound healing: a review on the harvesting systems, the ultrastructure of the graft and the mechanism of wound healing.
      A comparative proteomic study of human skin suction blister fluid from healthy individuals using immunodepletion and iTRAQ labeling
      • Müller A.C.
      • Breitwieser F.P.
      • Fischer H.
      • Schuster C.
      • Brandt O.
      • Colinge J.
      • et al.
      A comparative proteomic study of human skin suction blister fluid from healthy individuals using immunodepletion and iTRAQ labeling.
      Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis
      • Hoetzenecker W.
      • Ecker R.
      • Kopp T.
      • Stuetz A.
      • Stingl G.
      • Elbe-Bürger A.
      Pimecrolimus leads to an apoptosis-induced depletion of T cells but not Langerhans cells in patients with atopic dermatitis.
      Suction blisters: ex vivoRe-epithelialization and immune cell behavior in an ex vivo human skin model.
      • Rakita A.
      • Nikolić N.
      • Mildner M.
      • Matiasek J.
      • Elbe-Bürger A.
      Re-epithelialization and immune cell behaviour in an ex vivo human skin model.
      Further characterization of suction blister device for separation of the epidermis from dermis in human skin.
      • Falabella R.
      Suction blister device for separation of viable epidermis from dermis.
      Production of blister in normal human skin in vitro by blister fluids from epidermolysis bullosa.
      • Manabe M.
      • Naito K.
      • Ikeda S.
      • Takamori K.
      • Ogawa H.
      Production of blister in normal human skin in vitro by blister fluids from epidermolysis bullosa.
      Suction blistering as a method for the separation of the epidermis from the dermis.
      • Blank I.H.
      • Miller O.G.
      A method for the separation of the epidermis from the dermis.
      A comparative study of suction blister epidermal grafting and automated blister epidermal micrograft in stable vitiligo.
      • Gao P.R.
      • Wang C.H.
      • Lin Y.J.
      • Huang Y.H.
      • Chang Y.C.
      • Chung W.H.
      • et al.
      A comparative study of suction blister epidermal grafting and automated blister epidermal micrograft in stable vitiligo.
      HeatDermal‒epidermal separation by heat.
      • Jian L.
      • Cao Y.
      • Zou Y.
      Dermal-epidermal separation by heat.
      Epidermal grafting for wound healing: a review on the harvesting systems, the ultrastructure of the graft, and the mechanism of wound healing.
      • Kanapathy M.
      • Hachach-Haram N.
      • Bystrzonowski N.
      • Connelly J.T.
      • O’Toole E.A.
      • Becker D.L.
      • et al.
      Epidermal grafting for wound healing: a review on the harvesting systems, the ultrastructure of the graft and the mechanism of wound healing.
      Rapid, high-quality, and epidermal-specific isolation of RNA from human skin.
      • Trost A.
      • Bauer J.W.
      • Lanschützer C.
      • Laimer M.
      • Emberger M.
      • Hintner H.
      • et al.
      Rapid, high-quality and epidermal-specific isolation of RNA from human skin.
      Milling cutterDevelopment, standardization, and testing of a bacterial wound infection model on the basis of ex vivo human skin.
      • Schaudinn C.
      • Dittmann C.
      • Jurisch J.
      • Laue M.
      • Günday-Türeli N.
      • Blume-Peytavi U.
      • et al.
      Development, standardization and testing of a bacterial wound infection model based on ex vivo human skin.
      Human skin separation methodsDermal‒epidermal separation methods: research implications.
      • Zou Y.
      • Maibach H.I.
      Dermal–epidermal separation methods: research implications.
      Localization of basement membrane components after dermal‒epidermal junction separation.
      • Woodley D.
      • Sauder D.
      • Talley M.J.
      • Silver M.
      • Grotendorst G.
      • Qwarnstrom E.
      Localization of basement membrane components after dermal-epidermal junction separation.
      Methods for the separation of epidermis from dermis and some physiologic and chemical properties of isolated epidermis.
      • Baumberger J.P.
      • Suntzeff V.
      • Cowdry E.V.
      Methods for the separation of epidermis from dermis and some physiologic and chemical properties of isolated epidermis.
      Sheets of pure epidermal epithelium from human skin.
      • Medawar P.B.
      Sheets of pure epidermal epithelium from human skin.
      Porcine skin, a replacement for human skin?Ex vivo human and porcine skin effectively model candida auris colonization, differentiating robust, and poor fungal colonizers.
      • Eix E.F.
      • Johnson C.J.
      • Wartman K.M.
      • Kernien J.F.
      • Meudt J.J.
      • Shanmuganayagam D.
      • et al.
      Ex vivo human and porcine skin effectively model Candida auris colonization, differentiating robust and poor fungal colonizers.
      Comparison of human and porcine skin.
      • Khiao In M.
      • Richardson K.C.
      • Loewa A.
      • Hedtrich S.
      • Kaessmeyer S.
      • Plendl J.
      Histological and functional comparisons of four anatomical regions of porcine skin with human abdominal skin.
      Porcine skin as a substitute for human skin.
      • Summerfield A.
      • Meurens F.
      • Ricklin M.E.
      The immunology of the porcine skin and its value as a model for human skin.
      Distribution of esterase activity in porcine ear skin and the effects of freezing and heat separation.
      • Lau W.M.
      • Ng K.W.
      • Sakenyte K.
      • Heard C.M.
      Distribution of esterase activity in porcine ear skin, and the effects of freezing and heat separation.
      Abbreviations: iTRAQ, isobaric tags for relative and absolute quantitation; NaCl, sodium chloride; NH4SCN, ammonium thiocyanate.
      1 Owing to space limitations, we were not able to include and acknowledge all the work of researchers who contributed to this field.
      Supplementary Table S2Equipment and Tools for Skin Sampling and Excision
      Equipment and ToolsSourceIdentifier
      Disposable biopsy punches (2-, 4-, 6-, and 8-mm diameter)Kai MedicalREF BP-20F/-40F/-60F/-80F
      Disposable safety scalpelAesculap AGREF BA822Su
      Dermatome, Acculon 3TiAesculap AG, B. BraunGA670
      Dermatome blades for Wagner Dermatome, sterileAesculap AGGB228R/52377893
      Reaction tubes (1.5, 15, or 50 ml)Falcon, Greiner Bio-one, etc.REF 352098; catalog number 616 201
      Surgical scissors, tweezers
      Cooling packs for skin transportation
      Abbreviation: REF, reference.
      Supplementary Table S3Reagents Commonly Used for Disinfection of the Skin Surface
      ReagentsSourceIdentifier
      kodan forte farblosSchülke & Mayr26413-A/ 20000382-A
      DPBS, important for microbiome studiesGibco, Thermo Fisher ScientificREF 14200-067
      Sodium chloride solution (0.9%, 9 mg/ml, crucial for microbiome studies)B. Braun350 5731
      Sterile gauze (10 × 10 cm or smaller)HartmannREF 401 735
      Abbreviation: DPBS, Dulbecco’s PBS.
      Supplementary Table S4Reagents and Tools Applied for the Separation and Fixation of Skin Compartments Using Dispase
      Reagents and Tools for Skin SeparationSourceIdentifier
      Dispase II (neutral protease, grade II)Roche Diagnostics04942078001
      RPMIGibco, Thermo Fisher ScientificREF 31870-025
      DMEMGibco, Thermo Fisher ScientificREF 14200-067
      Penicillin‒streptavidinGibco, Thermo Fisher ScientificCatalog 15140122
      DPBS (1X, wash)Gibco, Thermo Fisher ScientificREF 14200-067
      Nitrile gloves
      Six-well plate/petri dish/reaction tube (1.5, 15, or 50 ml depending on the volume and size of the skin sample)
      Precision tweezers with sharp tips
      Pipettes and appropriate tips (10, 200, 1,000 μl)
      Reagents for Tissue FixationSourceIdentifier
      FA solution (7.5%, neutral, dilution in ddH2O)Liquid Production GmbHREF N-29401
      Acetone, Rotisolve ≥99.9%Carl RothT906.1
      Abbreviations: ddH2O, double-distilled water; DPBS, Dulbecco’s PBS; FA, formaldehyde; REF, reference.
      Supplementary Table S5Reagents and Tools Applied for the Separation of Skin Compartments Using NH4SCN
      ReagentsSourceIdentifier
      NH4SCNCarl Roth GmbH + Co. KG (Karlsruhe, Germany)CAS No. [1762-95-4] EG-Nr. 217-175-6
      DPBS (1X, diluent)Gibco, Thermo Fisher ScientificREF 14200-067
      Six-well plate/petri dish/reaction tube (1.5, 15, or 50 ml depending on the volume and size of the skin sample)
      Pipettes and appropriate tips (10, 200, 1,000 μl)
      Precision tweezers with sharp tips
      Abbreviations: CAS, chemical abstracts service; DPBS, Dulbecco’s PBS; NH4SCN, ammonium thiocyanate; No., number; REF, reference.
      Supplementary Table S6Reagents and Tools Applied for the Separation of Skin Compartments Using NaCl
      ReagentsSourceIdentifier
      NaClMERCKK37303004 719
      ddH2O (diluent)B. Braun201238001
      Six-well plate/petri dish/reaction tube (1.5, 15, or 50 ml depending on the volume and size of the skin sample)
      Pipettes and appropriate tips (10, 200, 1,000 μl)
      Precision tweezers with sharp tips
      Abbreviation: ddH2O, double-distilled water; NaCl, sodium chloride.
      Supplementary Table S7Reagents and Tools Applied for the Separation of Skin Compartments Using EDTA
      ReagentsSourceIdentifier
      UltraPure EDTA (0.5 M, pH 8.0)Invitrogen, Thermo Fisher ScientificREF 15575-038
      ddH2O (diluent)B. Braun201238001
      Six-well plate/petri dish/reaction tube (1.5, 15, or 50 ml depending on the volume and size of the skin sample)
      Pipettes and appropriate tips
      Precision tweezers with sharp tips
      Abbreviations: ddH2O, double-distilled water; REF, reference.
      Supplementary Table S8Equipment, Tools, and Reagents Applied for the Separation of Skin Compartments Using Low Pressure for Skin Blister Initiation
      Devices, Tools, and ReagentsSourceIdentifier
      Suction deviceElectronic Diversities
      Skin disinfection (if required)kodan forte farblos, Schülke & Mayr104029; GTIN: 9088883523954
      Sterile gauze (10 × 10 cm or smaller)HartmannREF 401 735
      Fixation base (styrofoam pad from a styrofoam box wrapped in aluminum foil)
      Precision tweezers with sharp tips, Surgical scissors, Micro-Fine syringe plus 30 G needle
      Abbreviation: REF, reference.
      Supplementary Table S9Devices and Tools for the Separation of Skin Compartments Using a Milling Cutter
      Devices and ReagentsSourceIdentifier
      Milling cutter (ball-shaped, 6 mm)ProxxonNo. 28725
      Dental micromotor handpieceMarathon N7, TPC Advanced Technology (Diamond Bar, CA)
      Fixation base (styrofoam pad taken from a styrofoam box and wrapped in aluminum foil)
      Syringes and needles
      Abbreviation: No., number.
      Supplementary Table S10Devices and Reagents Applied for the Separation of Skin Compartments Using Heat
      Devices and ReagentsSourceIdentifier
      Water bath (ddH2O) at 60 °C
      Ice water bath (ddH2O)
      DPBS (1X, wash)Gibco, Thermo Fisher ScientificREF 14200-067
      Precision tweezers with sharp tips
      Abbreviations: ddH2O, double-distilled water; REF, reference.
      Supplementary Table S11Antibodies and Reagents Used in this Study
      Antibodies and ReagentsSourceIdentifierDilution/Incubation/Duration
      Rabbit polyclonal ab to collagen IVAbcamAb65861:100, 4 °C, ON
      PE-mouse α-human CD1aBD PharmingenCat.5558071:100, 4 °C, ON
      Rat monoclonal α-CD207-AF488Dendriticsclone 929F3.01

      Cat.DDX0362
      1:200, 4 °C, ON
      CD3 ab, α-human-APC, REAfinityMiltenyi Biotecclone REA1151

      Lot.5210707051
      1:100, 4 °C, ON
      S100 rabbit abDAKOCat.Z0311Ready to use, RT, 4 h
      SOX10 mouse abSanta Cruz BiotechnologyCat.sc-3656921:33, 4 °C, ON
      Vimentin ab, α-human ab, FITC, REAfinityMiltenyi Biotecclone REA409

      Lot.5210610631
      1:100, 4 °C, ON
      PGP9.5 mouse abBio-Rad LaboratoriesCat.7863-10041:250, 4 °C, ON
      Mouse mAb to alpha-smooth muscle actin [1A4]Abcamab7817

      Lot.GR3356520-3
      1:100, 4 °C, ON
      FITC, anti-human CD31BioLegendclone WM59

      Cat.303104
      1:100, 4 °C, ON
      anti-LYVE-1 rabbit abAcris GmbHDP3500PS1:100, 4 °C, ON
      DAPISigma-AldrichCat.D95421 min, RT
      Alexa Fluor 647 goat anti-mouse IgG1 (γ1)Life Technologies, Thermo Fisher ScientificREF A212401:500, RT, 1 h
      Alexa Fluor 546 goat anti-rabbit IgG (H+L)Life Technologies, Thermo Fisher ScientificREF A110101:500, RT, 1 h
      ProLong Gold antifade reagentInvitrogen, Thermo Fisher ScientificCat. P369301 drop
      Abbreviations: ab, antibody; APC, allophycocyanin; Cat, catalog; h, hour; min, minute; ON, overnight; PE, phycoerythrin; REF, reference; RT, room temperature.

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