Background: This review summarizes uses and new applications for dermatological research of in vitro culture models of human skin explants (HSEs). In the last decade, many innovations have appeared in the literature and an exponential number of studies have been recorded in various fields of application such as process culture engineering, stem cell extractions methodology, or cell-to-cell interaction studies under physiological and pathological conditions, wound-healing, and inflammation. Most studies also concerned pharmacology, cosmetology, and photobiology. However, these topics will not be considered in our review. Summary: A better understanding of the mechanisms driving intercellular relationships, at work in the maintenance of 3D tissue architectures has led to the improvement of cell culture techniques. Many papers have focused on the physiological ways that govern in vitro tissue maintenance of HSEs. The analysis of the necessary mechanical stress, intercellular and cell-matrix interactions, allows the maintenance and prolonged use of HSEs in culture for up to 15 days, regardless of the great variability of study protocols from one laboratory to another and in accordance with the objectives set. Because of their close similarities to fresh skin, HSEs are increasingly used to study skin barrier repair and wound healing physiology. Easy to use in co-culture, this model allows a better understanding of the connections and interactions between the peripheral nervous system, the skin and the immune system. The development of the concept of an integrated neuro-immuno-cutaneous system at work in skin physiology and pathology highlighted by this article represents one of the new technical challenges in the field of in vitro culture of HSE. This review of the literature also reveals the importance of using such models in pathology. As sources of stem cells, HSEs are the basis for the development of new tissue engineering models such as organoids or optical clearing tissues technology. This study identifies the main advances and cross-cutting issues in the use of HSE.

Review Methodology

The model of human skin explants (HSEs) described in this review is often a tool included in broader research objectives. We selected all the elements referring to the use of HSEs in the Material and Method section from a very large number of studies covering a wide range of dermatological topics. Each paragraph refers to a fundamental area of dermatology: laboratory protocols, skin barrier function, wound healing, neurodermatology, inflammation, skin stem cells, and the future of the model. The main advances and important cross-cutting questions identified in the study are summarized in Table 1. The questions answered by the table are as follows. How do the HSEs change over the course of the culture? How to prolong the duration and quality of HSE tissues in culture? How to modify and assess the efficiency of the skin function? How to enhance skin wound healing? What are the new applications and advances made possible by the study of HSEs in psoriasis and atopic dermatitis? What types of stem cells can be extracted from HSEs? How to enrich the HSEs model?

Table 1.

Main cross-cutting questions posed by the review and their answers provided by citations from the manuscript

Cross-cutting issuesAnswers from the main results of the studiesAuthors
How do the HSEs change over the course of the culture? - During the first week of culture there is a production of antioxidant agents Vostálová et al. [1] 2019 
- After 8 days, TNFα induce type 1 collagen degradation through MMP1 Ågren et al. [2] 2015 
- After 9 days, TNFα and IL1 α induce arrest of 18 s transcription Neil et al. [3] 2020 
- HLA II-positive cells exist in burned HSEs revealing a real level of skin immunocompetence even when the skin is explanted Gross-Amat et al. [4] 2020 
How to prolong the duration and quality of HSE tissues in culture? - Proliferating keratinocytes exist in HSEs up to 75 days Frade et al. [5] 2015 
- Culture medium pH should be (>5.5), isotonic with skin cells and should contain 0.5 g L-1 of glucose. Fresh HSEs should not be stored at 4°C before culture Tarnowska et al. [6] 2019 
- Simple platelet-rich plasma improves skin regeneration Nicoletti et al. [7] 2019 
- HSEs require incubators or topical of humectant to rehydrate during culture Osseiran et al. [8] 2018 
- Eicosapentaenoic acid modifies the ceramide profile of HSEs and contributes to a skin anti-inflammatory function Kendall et al. [9] 2017 
- Vitamin C increases epidermal thickness, promotes collagen III production, and increases hyaluronic acid Gref et al. [10] 2020 
- Co-cultured neurons with HSE increase significantly epidermal thickness and cell density and decrease the rate of apoptosis Lebonvallet et al. [11] 2013 
- Lipopolysaccharids induce upregulations of IL-6, IL-8, TNFα, and IL-1. IL-10 in HSEs Gvirtz et al. [12] 2020 
How to modify and assess the efficiency of the skin function? - By pressure with a pad soaked in glue until most of the stratum corneum has been removed Danso et al. [13] 2015 
- In performing linear wounds that provide 50% less variable data than circular wounds Rizzo et al. [14] 2012 
- By specific detection of skin cells-interactive thermoreactive nanogels after alteration of the skin barrier Rancan et al. [15] 2017 
- By characterization of the structure of the stratum corneum and its chemical content by coherent Raman scattering imaging Osseiran et al. [8] 2018 
- By tracing epidermal cells with a fluorescent dye to obtain a real-time assessment of re-epithelialization course Nasir et al. [16] 2019 
- Thank to Hyperion technology that makes possible to assess all junctional proteins, nerve terminal localization, and neuromediator secretion Veenstra et al. [17] 2021 
How to enhance skin wound healing? - IL-10 significantly decreases epithelial cleft, increases epithelial height and neovascularization Balaji et al. [18] 2014 
- Mineralocorticoid antagonists overcome the negative impact of glucocorticoids on skin healing Nguyen et al. [19] 2016 
- CGRP, SP, and VIP promote cell proliferation in injured HSEs Cheret et al. [6] 2019 
- Co-cultured sensory neurons directly promote the healing of HSEs Cheret et al. [20] 2014 
What are the new applications and advances made possible by the study of HSEs in psoriasis and atopic dermatitis? - The new development of floating HSEs in a 32°C culture medium to mimic the initial events after skin barrier breakdown in atopic dermatitis Bauer et al. [21] 2021 
- TNFα and IL-17 act on skin ultrastructure and epidermal Langerhans cell number in psoriasis, resulting in an early alteration of skin defenses in psoriasis Donetti et al. [22] 2014 
- In atopic dermatitis, S. aureus contributes to the disruption of the skin barrier allowing the penetration of antigens into the epidermis Al Kindi et al. [23] 2021 
- Increase of TSLP release is associated with the activation of resident Langerhans cells and activation of AP-1 family during atopic dermatitis Bauer et al. [21] 2021 
What types of stem cells can be extracted from HSEs ? - Dermis-derived mesenchymal stem cells can be extracted from foreskin HSEs Park et al. [24] 2015 
- HSE-derived multipotent stromal cells can differentiate into osteoblastic lineage, adipocyte lineage, and endothelial lineage Vishnubalaji et al. [25] 2012 
- SKPs can be extracted from HSEs Bataille et al. [26] 2020 
- SKPs can be differentiated into peripheral neurons 
How to enrich the HSEs model? - By the development of complex co-culture models of neurons and HSEs Lebonvallet et al. [11] 2013 
- By the development of co-culture of trigeminal ganglion explants with HSEs to allow the formation of Merkel cell-neurite complexes Ishida et al. [27] 2020 
- By detection of epicutaneous infection of HSEs with S. aureus expressing green fluorescent protein (GFP) Popov et al. [28] 2014 
- By the development of a model of burned skin HSEs to evaluate the development of candidiasis Von Muller et al. [29] 2020 
- By the development of ex vivo human skin culture of partial thickness Xu et al. [30] 2012 
- By the development of HSEs combining normal keratinocytes and keloid fibroblasts Limandjaja et al. [31] 2020 
- By the development skin organoids engineering Lee et al. [32] 2020 
- By the technique of optical clearing of HSEs Cicchi et al. [33] 2005 
Cross-cutting issuesAnswers from the main results of the studiesAuthors
How do the HSEs change over the course of the culture? - During the first week of culture there is a production of antioxidant agents Vostálová et al. [1] 2019 
- After 8 days, TNFα induce type 1 collagen degradation through MMP1 Ågren et al. [2] 2015 
- After 9 days, TNFα and IL1 α induce arrest of 18 s transcription Neil et al. [3] 2020 
- HLA II-positive cells exist in burned HSEs revealing a real level of skin immunocompetence even when the skin is explanted Gross-Amat et al. [4] 2020 
How to prolong the duration and quality of HSE tissues in culture? - Proliferating keratinocytes exist in HSEs up to 75 days Frade et al. [5] 2015 
- Culture medium pH should be (>5.5), isotonic with skin cells and should contain 0.5 g L-1 of glucose. Fresh HSEs should not be stored at 4°C before culture Tarnowska et al. [6] 2019 
- Simple platelet-rich plasma improves skin regeneration Nicoletti et al. [7] 2019 
- HSEs require incubators or topical of humectant to rehydrate during culture Osseiran et al. [8] 2018 
- Eicosapentaenoic acid modifies the ceramide profile of HSEs and contributes to a skin anti-inflammatory function Kendall et al. [9] 2017 
- Vitamin C increases epidermal thickness, promotes collagen III production, and increases hyaluronic acid Gref et al. [10] 2020 
- Co-cultured neurons with HSE increase significantly epidermal thickness and cell density and decrease the rate of apoptosis Lebonvallet et al. [11] 2013 
- Lipopolysaccharids induce upregulations of IL-6, IL-8, TNFα, and IL-1. IL-10 in HSEs Gvirtz et al. [12] 2020 
How to modify and assess the efficiency of the skin function? - By pressure with a pad soaked in glue until most of the stratum corneum has been removed Danso et al. [13] 2015 
- In performing linear wounds that provide 50% less variable data than circular wounds Rizzo et al. [14] 2012 
- By specific detection of skin cells-interactive thermoreactive nanogels after alteration of the skin barrier Rancan et al. [15] 2017 
- By characterization of the structure of the stratum corneum and its chemical content by coherent Raman scattering imaging Osseiran et al. [8] 2018 
- By tracing epidermal cells with a fluorescent dye to obtain a real-time assessment of re-epithelialization course Nasir et al. [16] 2019 
- Thank to Hyperion technology that makes possible to assess all junctional proteins, nerve terminal localization, and neuromediator secretion Veenstra et al. [17] 2021 
How to enhance skin wound healing? - IL-10 significantly decreases epithelial cleft, increases epithelial height and neovascularization Balaji et al. [18] 2014 
- Mineralocorticoid antagonists overcome the negative impact of glucocorticoids on skin healing Nguyen et al. [19] 2016 
- CGRP, SP, and VIP promote cell proliferation in injured HSEs Cheret et al. [6] 2019 
- Co-cultured sensory neurons directly promote the healing of HSEs Cheret et al. [20] 2014 
What are the new applications and advances made possible by the study of HSEs in psoriasis and atopic dermatitis? - The new development of floating HSEs in a 32°C culture medium to mimic the initial events after skin barrier breakdown in atopic dermatitis Bauer et al. [21] 2021 
- TNFα and IL-17 act on skin ultrastructure and epidermal Langerhans cell number in psoriasis, resulting in an early alteration of skin defenses in psoriasis Donetti et al. [22] 2014 
- In atopic dermatitis, S. aureus contributes to the disruption of the skin barrier allowing the penetration of antigens into the epidermis Al Kindi et al. [23] 2021 
- Increase of TSLP release is associated with the activation of resident Langerhans cells and activation of AP-1 family during atopic dermatitis Bauer et al. [21] 2021 
What types of stem cells can be extracted from HSEs ? - Dermis-derived mesenchymal stem cells can be extracted from foreskin HSEs Park et al. [24] 2015 
- HSE-derived multipotent stromal cells can differentiate into osteoblastic lineage, adipocyte lineage, and endothelial lineage Vishnubalaji et al. [25] 2012 
- SKPs can be extracted from HSEs Bataille et al. [26] 2020 
- SKPs can be differentiated into peripheral neurons 
How to enrich the HSEs model? - By the development of complex co-culture models of neurons and HSEs Lebonvallet et al. [11] 2013 
- By the development of co-culture of trigeminal ganglion explants with HSEs to allow the formation of Merkel cell-neurite complexes Ishida et al. [27] 2020 
- By detection of epicutaneous infection of HSEs with S. aureus expressing green fluorescent protein (GFP) Popov et al. [28] 2014 
- By the development of a model of burned skin HSEs to evaluate the development of candidiasis Von Muller et al. [29] 2020 
- By the development of ex vivo human skin culture of partial thickness Xu et al. [30] 2012 
- By the development of HSEs combining normal keratinocytes and keloid fibroblasts Limandjaja et al. [31] 2020 
- By the development skin organoids engineering Lee et al. [32] 2020 
- By the technique of optical clearing of HSEs Cicchi et al. [33] 2005 

VIP, vasoactive intestinal peptide.

Update About the Maintenance of HSE, Focused on the Evaluation of Skin Degeneration Processes during Culture

HSEs are skin samples taken from human skin according to legal restrictions. Skin samples can be dissociated into different cell types. HSEs can be cultured and divided into epidermis and dermis or used in their entirety to obtain full thickness HSEs. HSEs represent a relevant alternative to animal models [34‒37].

There are several methods for culturing skin explants, such as trowel-type emerging skin explant cultures, well skin explants,floating skin explants, and submerged skin explants [38, 39]. More recently, Peramo et al. [40] developed a new sequential culture procedure. After being submerged for 10 days, skin explants were cultured at the air-liquid interface. Analysis of the cellular organization showed an additional epidermal layer including a horny layer, superimposed on the other. This experiment demonstrated the direct impact of the environment in the architecture of the skin. To better assess skin architectural alterations and to study the pathophysiological processes involved in explant aging, HSEs should be maintained in culture for a sufficient period of time. Frade et al. [5] sought to broadly extend the viability of HSEs. They observed proliferating keratinocytes up to 75 days. After this time, the skin thinned even though the dermal-epidermal junctions were preserved, and epidermal proliferation was maintained. The development of a medium capable of maintaining the viability of HSE in vitro becomes a new challenge. Tarnowska et al. [6] concluded that the medium should maintain a physiological pH (>5.5). It should be isotonic with skin cells and contain at least 0.5 g L-1 of glucose. They also showed that storing fresh HSEs overnight at 4°C altered metabolic activity and compromised the future of the skin cultures. Preservation of HSEs in vitro could be better achieved by the addition of platelet-rich plasma, which modulates epithelial cell viability in a model of injured HSE [7]. Simple platelet-rich plasma improves skin regeneration. However, the lack of accurate determination of plasma composition is the greatest drawback of using such a supplement to culture media.

The in vitro extension of HSEs allows to evaluate the process of skin degeneration. Vostálová et al. [1] revealed changes in the expression of inflammatory markers and in the production of antioxidant agents (including superoxide dismutase-2 and glutathione S-reductase) during the first week of culture of HSEs. These changes in inflammation and oxidative stress may represent the major limitation to long-term maintenance of HSEs in culture. Ågren et al. [2] specifically studied the effects induced by the addition of tumor necrosis factor α (TNFα), a well-known skin inflammatory marker, on collagen degradation after 8 days of culture. They showed that TNFα induced an increase in MMP-1 activity, explaining the progressive loss of type 1 collagen in HSEs. In terms of morphological changes and functional consequences in prolonged culture of HSEs include changes in gene expression levels according to E. Neil et al. [3]. During 20 days of culture, they found a marked arrest of 18 s transcription on day 9. They highlighted the difference between HSE and in vivo conditions with activation of interleukin 1 α (IL1 α) and TNFα. Histological architectural changes (parakeratosis or epidermal/dermal separation) were associated with modulation of skin barrier function.

At the interface between the body and the environment, the epidermis establishes a protection against environmental aggressions and water loss. The skin barrier that carries out this protection is a complex architectural system with variable permeability. In HSEs that maintain a 3D architecture in culture, the barrier capacities provided by the stratum corneum and intercellular junctions seem to be preserved in vitro and therefore assessable. Functional analysis tools of skin permeability such as transepithelial resistance and transepidermal water loss can be adapted to HSE in vitro. HSEs are increasingly used to study the physiology of skin barrier repair.

Alteration of the skin barrier can be performed experimentally as described by Danso et al. [13]. These authors used HSEs subjected to repeated pressure from a steel cylinder on which cyanoacrylate was spread and immediately applied to the skin until the stratum corneum had been removed. This model allows comparison of the effects of drug delivery across an intact or disrupted skin barrier as shown by Rancan et al. [15]. They identified cells interacting with thermoreactive nanogels after alteration of the skin barrier. The thermoreactive nanogels were labeled and loaded with fluorescein. In barrier-disrupted skin, after 2 h, only a small cluster of nanogel-positive Langerhans cells was found in the epidermis. In the dermis, a significant number of positive cells were found. These were mainly antigen-presenting cells. In barrier-disrupted skin diseases, the challenge remains to assess the ability to specifically reach immune cells in the deep dermis.

HSEs have also been used to explore the effect of the environment on the stratum corneum. Haftek et al. [41] introduced the new concept of stratum corneum “memory.” Cornification from the upper band of living keratinocytes is a rapid process involving intercellular junctions trapped at the cell periphery. Comparative analysis of these tight junction-like (TJ-like) structures in skin diseases has provided insight into cornification processes. The authors objectively found a significant overexpression of TJ-like in the stratum corneum, revealing an increase in the cohesion of the stratum corneum. Osseiran et al. [8] further characterized stratum corneum structure and chemical content by coherent Raman scattering imaging on HSEs subjected to different culture environments. HSEs underwent dehydration and humectant-induced rehydration. They were allowed to dry under ambient conditions before being moistened with a topical treatment of glycerin or hyaluronic acid. HSEs were then either maintained under ambient conditions or placed back in an incubator. Imaging and corneometer data showed that corneocytes in HSEs did not retain the hydration restored by humectants. The HSEs required the incubators and/or topical application of humectant to rehydrate. These results may be useful in evaluating ex vivo skin hydration restoration involved in skin barrier maintenance.

In addition, the ceramide profile in the skin seems to be a determining factor in the establishment of a solid lipid barrier. Van Smeden et al. [42] described a method that allows analysis of all known stratum corneum ceramide subclasses. The ceramide regulatory pathway was also studied by Kendall et al. [9]. They wanted to show whether eicosapentaenoic acid and docosahexaenoic acid could affect the profile of ceramides involved in the regulation of skin functions. HSEs were cultured for 6 days and supplemented with fatty acids. It seems that eicosapentaenoic acid could modify the ceramide profile and contribute to an anti-inflammatory function of the skin.

To strengthen the skin barrier, Gref et al. [10] studied the role of vitamin C in promoting epidermal thickness by modulating collagen. In this study, squalene was used to bind vitamin C to impart lipophilicity. Vitamin C bound to squalene increased epidermal thickness. It promoted collagen III production in HSE after only 10 days. Vitamin C positively regulates metalloproteinase expression and increases hyaluronic acid. It could be added in HSEs culture to maintain skin trophicity.

From the Restoration of the Skin Barrier to the Dynamics of Healing

The healing process is closely linked to the restoration of the skin barrier. A new imaging technique developed by Nasir et al. [16] revealed the dynamics of re-epithelialization in HSE. They traced epidermal cells with a fluorescent cell dye to obtain a real-time assessment of re-epithelialization. Re-epithelialization of human wounds ex vivo follows a collective migration of keratinocytes. It is possible to quantify cell movements and differentiation in ex vivo wound healing in a longitudinal manner. Li et al. [43] reveal differentiation into myofibroblasts of undifferentiated keratinocytes that have been grafted into granulation tissue. They used isolated epidermal sheets negative for vimentin and smooth muscle alpha actin (αSMA) and positive for E-cadherin. After transplantation in vivo, cells positive for vimentin or αSMA increased. E-cadherin-positive cells significantly decreased. Keratinocytes may transdifferentiate into myofibroblast-like cells in the wound.

All these studies must be carried out on previously validated skin wound models. Rizzo et al. [14] compared ex vivo linear and circular human wounds. They found that measurement on linear wounds provided 50% less variable data than those obtained from circular wounds. To complete the picture, Xu et al. [30] attempted to demonstrate that another model, called ex vivo culture of partial-thickness human skin, could represent a relevant means of assessing the skin healing process. This model includes the basal layer, the suprabasal layer, and the stratum granulosum. Up to day 10 of culture, the explants showed an increase in epidermal thickness and keratinocyte differentiation also progressed. Ex vivo human skin of partial thickness represents an intermediate model between keratinocyte culture and the complex architectural model of the HSE. The limitation of cell-cell interactions in this simpler model allows good segregation of the function of each cell type in the wound-healing molecular cascade.

At molecular level, Balaji et al. [18] suggested that IL-10 may enhance skin wound healing. They used a full thickness HSE on which wounds were created. Treatment with IL-10 resulted in a significant decrease in epithelial cleft, an increase in epithelial height, and an increase in vascular endothelial growth factor and neovascularization. While IL-10 appears to be an effector of skin wound healing, re-epithelialization of pathological skin wounds also appears to be enhanced by mineralocorticoid receptor antagonism. It is well established that glucocorticoids added to cultured HSEs induce delayed wound closure. Nguyen et al. [19] promoted the use of mineralocorticoid antagonists and highlighted its beneficial effects on pathological skin by overcoming the negative impact of glucocorticoids.

In contrast to the simple model developed by Xu et al. [30], a study on skin healing performed in a complex model associating injured HSEs and neurons in co-culture was conducted by Cheret et al. [6]. The authors aimed to test the effect of neuropeptides on skin wound healing. The involvement of vasoactive intestinal peptide, calcitonin gene-related peptide (CGRP), and substance P (SP) was tested. It appears that CGRP, SP, and vasoactive intestinal peptide promote cell proliferation in injured HSEs. It appears that SP and CGRP promote the production of collagen types 1 and 3. They observed an increase in metalloproteinase 2 (MMP-2) and metalloproteinase 9 (MMP-9) activity. At the cellular level, neuropeptides promoted adhesion of human dermal fibroblasts and differentiation of fibroblasts into myofibroblasts.

To complete our overview of wound healing, we feel it is important to look at pathological wound healing. Combining various intercellular-mediated abnormalities, keloid tissue suffers from a poor understanding [31]. Keloid formation involves disruptions in mediation between keratinocytes, endothelial fibroblasts, nerves, and immune cells. HSEs combining normal keratinocytes and keloid fibroblasts allow the study of the contribution of keloid fibroblasts to keloid formation. This model revealed intrinsic abnormalities in keratinocytes and keloid-derived fibroblasts. It identified unknown aspects of keloid behavior that could not be inferred from ex vivo biopsy analysis.

Skin-Nerves Interplay in Skin Homeostasis

In vitro tissue maintenance relies on a good understanding of the connections between the peripheral nervous system, the skin, and the immune system [44, 45]. Lebonvallet et al. [11], using a complex co-culture model of neurons and HSEs, showed a cross effect of HSE on neurite growth, and a re-innervation effect of HSE acting on the epidermis [11, 46]. In this model, only 10 days of co-culture were sufficient to establish a rich network of nerve fibers reinnervating the epidermis. While the neurite length of neurons, co-cultured with HSE, increased significantly after 6 days, epidermal thickness, cell density was higher in skin explants when associated with a rich nerve network. The rate of apoptosis was lower in skin explants when they were co-cultured with sensory neurons. The work of Cheret et al. [20] showed that sensory neurons directly promoted the healing of HSEs by increasing fibroblast and keratinocyte proliferation and the enzymatic activities of MMP-2 and MMP-9.

The cellular process of co-culture can be extended to other cell lines to further investigate cell-to-cell interaction. This method was developed by Ishida et al. [27]. Two methods of culturing mustache pads and trigeminal ganglion explants were combined in a co-culture model. They allowed the formation of Merkel cell-neurite complexes. The impact of sensory neurons on mast cells ex vivo in HSE was evaluated by Cheret et al. [47]. In re-innervated skin, mast cell numbers, maturation, and proliferation were significantly higher. These data suggest a high level of inter-communication between neurons and other cell types in the skin.

These co-culture models have been used to test transcutaneous neuronal activation and neuroinflammation. Lebonvallet et al. [48] using a patch-clamp technique on neuronal fibers observed repetitive electrical spikes in response to the application of capsaicin directly to HSEs [48, 49]. Lestienne et al. [50] observed that the addition of an SP inhibitor on HSEs inhibited the release of pro-inflammatory cytokines. The authors of these papers demonstrated that HSEs could be used for multiple functional investigations involving cell-cell interactions.

Co-culture models of HSEs and neurons allow for longer maintenance of the epidermis and limit the degeneration process. The future of neuron/HSEs co-cultures could be based on enhancing nerve-skin interactions. By mimicking the normal interaction between skin and nerves, this model could help understand the pathological process that leads to psoriasis, atopic dermatitis, and sensitive skin where the change in neurite density is associated with the modulation of skin trophicity [51, 52]. It could allow the identification of new therapeutic targets to treat more effectively “neuro-dermatological pathologies.” These models have already shown their advantages, in particular by the in vitro visualization of “synapse like” neurocutaneous structures [53].

Cell-to-Cell Interplay in Skin Pathology

There is a large body of work investigating the role of cytokines in inflammation in HSEs. About that, Gvirtz et al. [12] evaluated the secretion profile of key cytokines upon lipopolysaccharide stimulation. They observe upregulations of IL-6, IL-8, TNFα, and IL-1. IL-10, an anti-inflammatory cytokine, was also induced by lipopolysaccharide.

First, the variety of in vitro skin models makes it necessary to guide researchers on the most relevant choice. That is the reason why Desmet et al. [54] published a review critiquing the different types of skin culture models in psoriasis. Psoriasis is the most common area of research in dermato-immunology that includes studies on HSEs. Prignano et al. [55] studied the effect of TNFα and IL-17 on ultrastructure, immunophenotype, and epidermal Langerhans cell number in psoriasis HSEs [22, 55]. They observed like Donetti et al. [22] that IL-17 and TNFα could induce an early alteration of skin defenses directly inhibiting keratinocyte proliferation. They also showed a link between inflammation and skin barrier function using occludin immunostaining. To complete the molecular aspect, Gallais Sérézal et al. [56] explored whether T-cell activation induces different tissue response patterns in healthy and psoriatic skin. In this paper, the authors showed a correlation between immune activation and tissue remission time. It appears possible to individually predict disease relapse from individual tissue responses to IL-17A-directed T-cell stimulation [56, 57].

Second, skin barrier function, skin colonization by Staphylococcus aureus, and specific immunologic characteristics of the skin are closely related in atopic dermatitis. In this way, HSEs can be seen as a cross-disciplinary model for a better understanding of pathophysiology. Al Kindi et al. [23] explore the type 2 immune response to S. aureus in atopic dermatitis. Using atopic HSEs, they demonstrated a link between S. aureus secretome and IL-33 release. HSEs were exposed to S. aureus supernatant. The second immunoglobulin-binding protein of S. aureus, which promotes the TH2 response, also contributes to the disruption of the skin barrier. This mechanism allows the penetration of antigens into the epidermis, leading to the initiation of host type 2 immune responses. To complete, Van Drongelen et al. [58] described the ex vivo evolution of HSE from healthy skin of patients with atopic dermatitis with different cellular genotypes of the filaggrin gene. They found that mutations in the filaggrin gene did not affect the growth of HSEs in vitro. In vitro HSEs retain their filaggrin genotype-phenotype. They confirmed that the expression of most proteins remains similar to in vivo conditions.

Finally, Bauer et al. [21] usedfloating HSEs in a 32°C culture medium to mimic the initial events after skin barrier breakdown in atopic dermatitis. They observed an increase in the release of stromal thymic lymphopoietin (TSLP) associated with the activation of resident Langerhans cells. The authors also describe the activation of transcription factors of the activator protein-1 (AP-1) family during atopic dermatitis. This transcription factor is involved in the production of TSLP and highlights a new therapeutic approach targeting early events of skin inflammation in atopic dermatitis [21].

The HSE model is not an exception to the recent trend in microbiota studies. Percoco et al. [59] conducted a study of HSEs subjected to microbial stimuli. They showed that S. epidermidis and P. fluorescens affected the structure of skin explants without penetrating the living epidermis. The morphology of these HSEs changed from h72 and h96, especially in the basal stratum. To get a better understanding of the alteration of skin structure mediated by bacterial infections, Popov et al. [28] induced epicutaneous infection of HSEs with S. aureus expressing green fluorescent protein (GFP). In this way, they were able to follow the bacteria during the colonization process. They found that immunization against S. aureus altered the behavior of the commensal bacterial population and protected against invasive infections. This modulation could be mediated by the aryl hydrocarbon receptor (AhR). In addition, Rademacher et al. [60] pointed out that blocking AhR in HSEs infected with S. epidermidis resulted in increased growth of S. epidermidis. These models of HSEs are also well suited to study skin infections such as superficial fungal infections (T. rubrum) [61, 62] or anaerobic skin infections [63].

To illustrate the wide application of HSE in the field of infection, Von Muller et al. [29] established a model of burned skin HSEs to evaluate the development of candidiasis. Wounds were infected with C. albicans with or without removal of the burned outer layer before infection. They show that the contact of fungal cells with the necrotic dermis allows a deeper penetration of the fungus into the damaged tissue. In the upper dermal layers, under the burned epidermis, large clusters of neutrophils appeared. This reveals the migration capabilities of neutrophils to the site of the infected wound. Surprisingly, C. albicans did not influence cytokine release. In vitro, HSEs maintain an active immune response and are able to counteract C. albicans infection. In a similar model [4], Gross-Amat et al. [4] found HLA II positive cells in burned HSEs. This very interesting result tends to prove a non-specific activation of immune or non-immune cells in HSEs. For us, this is one of the major discoveries of the last decade concerning in vitro co-culture of HSEs. These skin models show a real level of immunocompetence even when the skin is explanted. Other studies remain to be written regarding the immunocompetence of HSEs in vitro.

Focusing on the molecular level, Gonnet et al. [64] sought to determine the role of IL-32 produced by keratinocytes on surrounding Langerhans cells. The model used involves HSEs infected with modified vaccinia virus Ankara. The authors observe the ability of infected keratinocytes to produce IL-32 promoting the detachment of Langerhans cells allowing their migration. Thanks to the multicellular aspect of HSEs, they showed that IL-32 is a molecular link between keratinocytes and Langerhans cells. To complete, Bryden et al. [65] used analyses of HSEs to observe a local innate immune response to arbovirus inoculation. In their paper, they showed that only stromal cells and macrophages expressed the structural gene transcript of the virus. When hosts were treated prior to infection with a topical Toll-like receptor 7 agonist at the site of inoculation, local, and systemic progression of infection was suppressed.

The wide expansion of stem cell technology also applies to HSEs. They are a valuable and accessible source of highly proliferative stem cells [66, 67]. Initially, Park et al. [24] proposed to isolate and determine the proliferative capacity of dermis-derived mesenchymal stem cells from foreskin HSEs. Using an enzymatic method, the derived mesenchymal stem cells were isolated and maintained in culture. The adherent stromal cells exhibited fibroblast-like morphology. They were positive for markers associated with stromal cells and negative for markers of endothelial and hematopoietic lineages. In culture, they were able to differentiate into skin endothelial stromal cells. These multipotent cells were highly proliferative and grew without additional growth factors. These stem cells retained the ability to differentiate into several cell lineages, including osteocytes, chondrocytes, adipocytes, and hepatocytes.

Then, to further characterize the stem cells extracted from HSEs, Vishnubalaji et al. [25] compared HSE-derived multipotent stromal cells with bone marrow- and adipose tissue-derived mesenchymal stem cells. Their potential to differentiate into osteoblastic lineage, adipocyte lineage, and endothelial lineage has been certified by immunophenotyping, flow cytometry, and immunofluorescence. In addition to being an accessible source of highly proliferative stem cells, HSEs represent a stem cell donor for tissue regeneration.

Recently, Bataille et al. [26] obtained sensory neurons from HSEs in vitro. After different in vitro differentiation steps, skin-derived precursors (SKPs) extracted from HSEs were characterized as neural crest-derived neural progenitors by immunostaining and quantitative polymerase chain reaction. At the end of the differentiation process, SKPs showed a differentiation profile of peripheral neurons. They expressed sodium, calcium, and potassium channels that were active during patch-clamping. Peripheral neurons obtained simply from SKPs extracted from HSEs could be very interesting to avoid the use of embryonic stem cells, artificial stem cells, animal neurons, or to replace expensive industrial stem cells.

For us, the future of skin models lies in the engineering of skin organoids. Lee et al. [32] found a way to grow skin organoids in vitro from human pluripotent stem cells. After 5 months, they obtained a skin organoid composed of a stratified epidermis, a fat-rich dermis, pigmented hair follicles, and sebaceous glands. This de-novo assembled model combined a network of sensory neurons with Schwann cells targeting Merkel cells. They prove that almost complete skin can be self-assembled in vitro.

Our review illustrates the increasing need for skin models to test biological aspects of skin physiology and therapeutics [49]. This increase goes hand in hand with the limitation of animal experiments. Another aspect of the future of dermatological research may lie in the 3D printing of skin organoids. This technology would allow the study of skin without the need for human skin, which may be in limited supply in scientific laboratories. The greatest strengths of HSEs remain in their ability to be enriched with other cell types. It gives insight into the great complexity of interactions that take place in the physiology of the skin.

We believe that, these models could also be analyzed in a single-cell transcriptomic assay to characterize the interactions between different cell types within the tissue. The study of cell-cell interaction performed on HSEs could be possible on the same microscopic section. With Hyperion technology, it is possible to simultaneously assess all junctional proteins, nerve terminal localization, and possibly neuromediator secretion [17].

Finally, the technology of optical clearing of samples could make the biological processes at work in skin homeostasis directly observable. Some studies already describe optical skin-clearing techniques using aqueous solvents. The objective was to increase the depth of acquisition in confocal or biphotonic microscopy by homogenizing the refractive index of the tissues. This technique deserves to be developed [33, 68].

At the end of this review, it appears that the study conducted on HSEs has grown exponentially, which was not the practice in 2010. The old culture protocols have been updated to extend the culture time of HSEs, allowing the emergence of new concepts. The HSE model is still adapted to the understanding of fundamental biological processes and has allowed the discovery of central actors of skin homeostasis concerning aging, skin barrier function, and wound healing such as ceramide interleukins, vitamin C, neuropeptides. Its 3D architecture allows the evaluation of cell-cell interactions, illustrated by the observation of immune cell migration in the skin layers and skin/neuron interaction, which is not the norm in many fields of applied biology. These experiments have led to the recent emergence of the concept of cutaneous neuro-immunoregulation. This model retains a degree of in vitro immunocompetence that is very interesting for the study of skin pathologies such as psoriasis or atopic dermatitis, which remain to be further investigated. It represents a very interesting source of stem cells to avoid the use of very expensive industrial cells. This article illustrates the great diversity of the use of skin models in dermatology and highlights the potential for discovery that remains. The mastery of this model represents an essential path for the development of new technologies such as skin organoids, optical clearing of tissue samples, or 3D printing of skin.

The aims of this work were to describe the main advances in the understanding of intercellular interactions allowed by the development of techniques for culturing and maintaining HSE in vitro and to detail the new fields of application opened up by these discoveries.

The authors have no conflicts of interest to declare.

This study did not receive any funding.

Ianis Cousin is the lead author of this review and the corresponding author. Nicolas Lebonvallet and Philine de Vries reviewed and supervised the manuscript. Laurent Misery is the director of the laboratory where researches are conducted.

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