The use of skin explants in Brazil: A scoping review of applications,methodologies and research trends

Abstract

The ex vivo Human Organotypic Skin Explant Culture (hOSEC) model utilises skin fragments, mainly obtained from plastic surgery procedures, as the primary source of material. The model is used for testing that requires human tissue, and as the basis for various research models. The objectives of the current study were to review, analyse and summarise the published literature on the use of ex vivo human skin explants in experimental studies in Brazil. The literature search was conducted within three databases, using terms related to explants and study location combined with defined eligibility criteria. Sixteen studies were considered eligible for further detailed analysis in the review. The geographical location of these studies was concentrated in the South-Eastern region of Brazil, and demonstrated a prevalence of explants obtained during plastic surgery procedures. Five main focus areas were identified in the studies, namely: disease pathology, cellular ageing, pharmacological testing of cosmetic ingredients, wound healing, and cell culture optimisation — with pathology-focused models being the most common. Despite the search strategy being capable of identifying the diverse characteristics of the studies, the research protocols used in the studies were heterogeneous. This is an intrinsic limitation of the published literature in general, which prevents direct comparisons and hinders reproducibility. Human skin explants were shown to represent versatile tools, with potential for expansion into other areas. The creation and dissemination of standardised methodologies and guidelines for human skin explant-based research are essential. This information, as well as the promotion of the use of such models, will contribute to a reduction in the use of animals in experiments.

Keywords

alternative models Brazil ex vivo human skin skin explant

Introduction

The hOSEC model is used in various research contexts, such as for testing topical medications on wounds, evaluating cosmetics and observing skin physiology under specific situations.⁴ The development of treatments for dermatological conditions such as melanoma and hypertrophic scars, which affect various parts of the skin, can benefit from the use of explants for analysing how the tissue responds to drugs, for example, in terms of toxicity and inflammatory modulation.^4–6 For the development of scar treatments, explants offer the advantage of preserving the 3D organisation of human skin, permitting the evaluation of processes such as re-epithelialisation and matrix remodelling.⁷ Therefore, the use of hOSEC models represents a promising strategy for developing and validating new treatments within a broad range of applications.

Globally, there are extensive efforts to reduce the use of animal models in research, which are supported by the national institutions in many countries, such as the US National Institutes of Health and respective regulatory agencies.⁸ Brazil has also implemented regulations aligned with the Three Rs principles (replacement, reduction and refinement), including the prohibition of animal testing for cosmetics, which was established by Law 14.198/2021 and consolidated by the 2023 resolutions of the National Council for the Control of Animal Experimentation (CONCEA). These regulations restrict the use of animals for the development and testing of cosmetic products and ingredients, when validated alternative methods are available.⁹

Despite its potential as an experimental model and a means of reducing animal use, there is an information gap relating to the extent of use of the hOSEC model for research purposes in Brazil, as well as the precise details of the studies in which it has been used. This information could serve as a starting point for the creation and dissemination of standardised methodologies and guidelines. In this context, identifying and understanding the use of hOSEC models in Brazil is essential to expanding their application in new areas of research, promoting access to specific funding, ensuring methodological replicability and contributing to a reduction in the use of animals in experiments. The current study aims to review, analyse and summarise the published literature on the use of ex vivo human skin explants in experimental studies conducted in Brazil.

Methods

This study consists of a scoping review addressing the use of ex vivo human skin explants in Brazil. The study used the methodology described in the PRISMA-SCR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guideline for scoping reviews.¹⁰ The study protocol was previously defined and registered on the Open Science Framework (OSF) platform under DOI: 10.17605/OSF.IO/NW5Z7.¹¹

Eligibility criteria for inclusion

There were four criteria applied during the selection of the retrieved studies for further detailed analysis in the review.

(1) Type of study: Experimental studies (ex vivo) using explanted human skin as a research model.

(2) Tissue source: Studies using human tissue obtained through elective surgical procedures or donation.

(3) Study location: Research conducted in Brazil, regardless of the nationality of the authors. The geographical location where data were collected, or the experiments were conducted, must be within Brazilian territory.

(4) Publication type: Articles published in peer-reviewed journals.

Search strategy and data sources

The search was conducted within three databases: (i) Medline, via PubMed; (ii) Embase; and (iii) the Virtual Health Library (VHL). Search terms related to explants (such as “skin explant”, “ex vivo model” or “human skin explant”) and study location (“Brazil”, “South America”) were used and formulated appropriately for each database. The final search strategies used for each database can be found in Table 1.

Table 1.

The search strategies used to retrieve papers for the review.

Database	Search strategy
Embase	(‘brazil'/exp OR ‘brazil’ OR ‘federative republic of brazil’ OR ‘united states of brazil’ OR ‘brazilian'/exp OR ‘brazilian’ OR ‘brazilians’ OR ‘brazilian institution’ OR ‘south america'/exp OR ‘south america’ OR ‘america, south’ OR ‘institution in brazil’) AND (‘skin explant'/exp OR ‘ex vivo skin’ OR ‘ex vivo model’ OR ‘human skin explant’ OR ‘skin model'/exp OR ‘ex vivo model of human skin’ OR ‘hosec’ OR ‘human organotypic skin explant’ OR ‘ex vivo skin cultures’ OR ‘ex vivo protocol’ OR ‘ex vivo skin protocol')
PubMed	((“Brazil”[Mesh]) OR “South America”[Mesh]) AND ((((Ex Vivo Skin) OR (Ex Vivo Model)) OR (hosec)) OR (Ex Vivo Protocol) OR (Skin explant))
Virtual Health Library (VHL)	((Explante de pele) OR (skin explant) OR (pele humana explantada) OR (human skin explant) OR (ex vivo skin model) OR (hosec) OR (hosec model) OR (cultura ex vivo de pele humana) OR (ex vivo human skin culture) OR (cultura ex vivo de pele) OR (ex vivo skin culture) OR (ex vivo skin culture model)) AND ((Brazil) OR (Brazilian Institute) OR (Brasil) OR (Instituto Brasileiro) OR (Hospital Brasileiro) OR (Brazilian Hospital))

Study selection and data extraction

Once the database search was completed, the data were transferred to Rayyan™ software (Rayyan Systems Inc., Cambridge, MA, USA) where they were evaluated by two ‘masked’ researchers, who conducted the evaluation independently. Disagreements were resolved by discussion and consensus with a third researcher. Studies that were incomplete, or did not meet the inclusion criteria, were excluded. After selecting the studies, the researchers collated the necessary data by using the Google Sheets platform.

Data analysis

The following fifteen variables were obtained: author; title; study design; geographical location of the study institution; ethics committee approval; study objectives; source of human skin; donor age; immediate conditioning of the explant; skin fragment collection method; fragment size; fragment thickness; culture medium characteristics; analyses performed; and summary of results.

The data analysis was conducted according to the following topics: geographical distribution of the studies; general focus area of the study; fragment characteristics; culture medium characteristics; and the analytical techniques used. The following categories were used for classifying the experimental studies, in terms of their culture duration: short-term (≤ 6 days), medium-term (7–30 days) and long-term (31–90 days).

Results

The searches were conducted within three databases: PubMed (n = 43), Embase (n = 181), and VHL (n = 11), identifying a total of 235 studies. Of these, ten were removed due to duplication before the screening stage. The remaining 225 articles were then screened, resulting in the exclusion of 198. In total, 4 full-text articles could not be found. Consequently, 23 were evaluated in full for eligibility. At this stage, the following exclusion criteria were applied: uncertain or inadequately described geographical location of the study (n = 4); and studies that did not address models of interest (n = 3). An illustration of the process is shown in Figure 1. At the end of this process, 16 studies were found to be eligible and were included in this scoping review. The 16 studies further analysed in this review are described in Table 2.

Figure 1.

Flow chart of the process involved in selecting the papers for the review.

Table 2.

Experimental details provided in the papers reviewed.

Article	Title	Culture conditions		Tissue culture setup
Article	Title	Medium	Supplements	Tissue culture setup
Gragnani A et al.¹²	Dimethylaminoethanol affects the viability of human cultured fibroblasts	Fibroblast medium	None	Submerged
Afornali A et al.¹³	Triple nanoemulsion potentiates the effects of topical treatments with microencapsulated retinol and modulates biological processes related to skin aging	DMEM	10% FBS + 1% antibiotics (penicillin + streptomycin)	Air–liquid interface
Frade MAC et al.³	Prolonged viability of human organotypic skin explant in culture method (hOSEC)	DMEM	10% FBS + 1% antibiotics (penicillin + streptomycin + amphotericin B) + ʟ-glutamine	Air–liquid interface
Peres NTA et al.¹⁴	In vitro and ex vivo infection models help assess the molecular aspects of the interaction of Trichophyton rubrum with the host milieu	Skin Graft Fluid	None	Air–liquid interface
D’Angelo Costa GM et al.¹⁵	Is vitamin D3 transdermal formulation feasible? An ex vivo skin retention and permeation	Non-standard (PBS)	Ethanol (50%)	Air–liquid interface (Franz diffusion cell)
Alcantara GP et al.¹⁶	Evaluation of ex vivo melanogenic response to UVB, UVA, and visible light in facial melasma and unaffected adjacent skin	DMEM	None	Submerged
Romana-Souza B et al.¹⁷	Topical application of a commercially available formulation of vitamin C stabilized by vitamin E and ferulic acid reduces tissue viability and protein synthesis in ex vivo human normal skin	DMEM	10% FBS + 1% antibiotics*	Air–liquid interface
Leite MN et al.¹	Ex vivo model of human skin (hOSEC) for assessing the dermatokinetics of the anti-melanoma drug Dacarbazine	DMEM	10% FBS + 1% antibiotic + antifungal solution	Air–liquid interface
Saguie BO et al.¹⁸	An ex vivo model of human skin photoaging induced by UVA radiation compatible with summer exposure in Brazil	DMEM	10% FBS + antibiotics*	Air–liquid interface
Eberlin S et al.¹⁹	Ex vivo human skin: An alternative test system for skin irritation and corrosion assays	DMEM	10% FBS + 0.1% gentamicin	Air–liquid interface
Weihermann AC et al.²⁰	Modulation of photoaging-induced cutaneous elastin: Evaluation of gene and protein expression of markers related to elastogenesis under different photoexposure conditions	Not applicable (RNA extraction)	None	Not applicable (RNA extraction)
Carlos ECS et al.²¹	Imiquimod-induced ex vivo model of psoriatic human skin via interleukin-17A signalling of T cells and Langerhans cells	DMEM	10% FBS + 1% penicillin + 1% streptomycin	Air–liquid interface
Pereira Oliveira CN et al.⁵	Nanoemulsions based on sunflower and rosehip oils: The impact of natural and synthetic stabilizers on skin penetration and an ex vivo wound healing model	DMEM	10% FBS + 1% antibiotics (penicillin + streptomycin + amphotericin B) + ʟ-glutamine	Air–liquid interface
Alencar-Silva T et al.²²	Screening of the skin-regenerative potential of antimicrobial peptides: Clavanin A, Clavanin-MO, and Mastoparan-MO	DMEM	10% FBS + 1% antibiotics (penicillin + streptomycin)	Air–liquid interface
De Paula NA et al.²³	Human skin as an ex vivo model for maintaining Mycobacterium leprae and leprosy studies	DMEM	10% FBS + 1% antibiotics (penicillin + streptomycin)	Air–liquid interface
Souto-Silva MV et al.⁶	Toxicity and dermatokinetic analysis of ibrutinib in human skin models	DMEM	10% FBS + 1% antibiotics (penicillin + streptomycin)	Air–liquid interface

*Not specified. DMEM = Dulbecco’s Modified Eagle Medium; FBS = fetal bovine serum; PBS = phosphate-buffered saline solution.

Geographical distribution of the studies

The South-Eastern region of Brazil stood out in terms of the amount of published research employing skin explants as ex vivo models that has been carried out. Twelve of the sixteen selected articles (75%) were from institutions located in this region,^{1,3,5,12,14–19,21,23} with nine of these twelve originating from institutions within the state of São Paulo.^{1,3,5,12,14–16,19,23} In addition, two projects were carried out in the Mid-West^6,22 and two (each corresponding to 12.5%) in the South of the country.^13,20

According to the National Code of Ethics, the use of this type of material (skin fragments) is considered as ‘research involving human subjects’. Therefore, the projects must be submitted to, and approved by, a local Research Ethics Committee. In addition, an informed and freely-obtained consent form from each participant must be obtained, in accordance with Brazilian legislation and the ethical principles established by the Declaration of Helsinki. All studies included in the current review were approved by a local Research Ethics Committee, except for one study that did not clearly report ethical evaluation.¹³

General focus area of the study

After analysing the articles in detail, five main focus areas in which the models had been used were identified, namely: disease pathology, cellular ageing, pharmacological testing of cosmetics ingredients, wound healing, and cell culture optimisation.

Disease pathology

Disease models involved using the ex vivo human skin tissue to replicate a specific disease environment, either for observational purposes or to evaluate a therapeutic intervention. The diseases and conditions studied in the reviewed articles were: melanoma,^1,6 melasma,¹⁶ leprosy,²³ psoriasis²¹ and dermatophytosis.¹⁴

Cellular ageing

These models were employed to visualise changes in tissue structures resulting from photoageing.^18,20

Pharmacological formulation testing

The models were used to test pharmacological ingredients for cosmetics, specifically: dimethylaminoethanol (DMAE),¹² retinol,¹³ and vitamin D₃¹⁵ and vitamin C.¹⁷

Wound healing

Models were used to evaluate the skin’s regenerative capacity in the presence of vegetable oil nanoemulsions⁵ and antimicrobial peptides.²²

Cell culture optimisation

Models were used to determine how to extend the useability window of the explant³ and promote the skin’s viability for irritation testing.¹⁹

Fragment characteristics

In 15 studies (93.75%), explants derived from plastic surgery procedures were used, with abdominoplasty being the most prevalent (50%).^{1,3,5,14,15,19,21,23} Other types of procedures were also reported, including: otoplasty (12.5%),^18,21 mammoplasty (18.75%),^3,5,20 rhytidoplasty (6.25%)²⁰, and blepharoplasty (6.25%).²⁰ Four studies (25%) did not specify the type of plastic surgery performed;^12,13,17,22 one study (6.25%) used facial skin from patients with melasma.¹⁶

Regarding collection of the skin fragments, the use of punch biopsy was observed in ten studies (62.5%)^{1,3,6,16–19,21–23} and the excision technique in six studies (37.5%).^5,12–15,20 The average diameter of the fragments collected by the first method was 6.7 mm, while for excision, the average cut edge size was 8.9 mm. All studies used full or partial skin thickness (epidermis and dermis).

Culture medium characteristics

The most commonly used medium for culturing the ex vivo tissue was Dulbecco’s Modified Eagle Medium (DMEM), which was used in twelve of the sixteen studies (75%).^{1,3,5,6,13,16–19,21–23} A Skin Graft Fluid (i.e. a liquid medium that contains human plasma, saline solution, and neomycin) was used for this purpose in one of the sixteen studies (6.25%),¹⁴ and phosphate-buffered saline (PBS)¹⁵ was similarly used in another single study (6.25%). Importantly, PBS is not a nutrient-containing medium but, rather, it is a chemical buffer solution for use in skin permeation tests. One article (6.25%) used a fibroblast medium, to isolate them,¹² and another (6.25%), which focused on RNA extraction, did not involve the culture of cells from explants.²⁰ From a supplementation perspective, 10% (v/v) fetal bovine serum (FBS) was used in 11 protocols (69%), indicating its wide acceptance as a means of supporting cell viability.^{1,3,5,6,13,17–19,21–23}

The culture media used in the studies often included antibiotics, mainly combinations of penicillin and streptomycin; this was evident in seven of the 11 papers that used antibiotics (64%).^{3,5,6,13,21–23} Two studies (12.5%) supplemented the medium with ʟ-glutamine^3,5 and another with 50% ethanol (6.25% each).¹⁵ Four studies (25%) did not specify the supplements used primarily to preserve the fragment.^12,14,16,20 As for the tissue culture setup, 13 protocols (81%) employed an air–liquid interface model, providing conditions close to those found in vivo,^{1,3,5,6,13–15,17–19,21–23} while two studies (12.5%) kept the explants submerged.^12,16 This information is described in Table 2. In six of the articles (38%), the tissues were kept viable in culture for a short period of time (≤ 6 days).^1,13–16,19 In one of these six articles (Eberlin et al.¹⁹), two shorter-term sub-experiments were performed, with different durations. In seven studies (43%), explant culture was maintained for between 7 and 30 days (average duration),^{5,6,12,17,18,21,22} except in Pereira Oliveira et al.,⁵ in which one of the experiments lasted 7 days (comparing nanoemulsifications) and the other lasted 14 days (to verify the viability of the ex vivo wound model). Long-term models (31–90 days) were described in two (12.5%) of the papers.^3,23 Only one article did not fit this approach.²⁰ With regard to changing the culture medium, in eight papers (50%) it was reportedly performed, between one and four times.^{1,5,6,12,17–19,21} Two articles (12.5%) made more than five changes,^17,23 given that, in Romana-Souza et al.¹⁷, the experiments were performed at three different time points (7, 10, and 13 days), this study could be classified in both categories. Finally, six studies (43.75%) did not specify or meet the frequency evaluation criteria.^{3,13–17,20,22}

Analytical techniques used

Within the five main focus areas, namely: disease pathology, cellular ageing, pharmacological testing of cosmetics ingredients, wound healing, and cell culture optimisation, the types of experiment carried out varied considerably. A summary of the analytical techniques used are outlined below and in Table 3.

Table 3.

A summary of the analytical techniques used in the reviewed articles.

Analytical techniques used	Study focus area
Analytical techniques used	Disease pathology	Cellular ageing	Pharmacological testing of cosmetics ingredients	Wound healing	Cellular culture optimisation
Histopathology and special stains	Haematoxylin and Eosin (HE),^1,21,23 Fontana–Masson (melanin),¹⁶ Masson’s trichrome (collagen)²¹	HE^18,20	HE¹⁷	HE⁵	HE³
Protein expression	Immunohistochemistry and im munofluorescence (PCNA, immune cells),²¹ Western blot,²¹ ELISA²¹	Immunohistochemistry (elastin),^18,20 Western blot (MMPs)¹⁸	Western blot (vitamin C),¹⁷ immunohistochemistry (cellular proliferation)¹⁷	Immunohistochemistry (cellular proliferation, Ki-67)⁵	Immunohistochemistry (Ck5/6, Ck10, Ki-67)³
Gene expression	RT-qPCR (immune response, pathogen–host interaction)^14,21,23	RT-qPCR (elastin, MMPs)²⁰	RT-qPCR (vitamin C, retinol)^13,17	RT-qPCR (Ki-67)²²	NA
Apoptosis and cell viability	Colorimetric tests (TTC),^1,6 (MTT)²¹	NA	Colorimetric tests (MTT)¹⁷, direct counting and flow cytometry¹²	NA	Colorimetric tests (MTT)¹⁹
Permeation and dermatokinetics	High Performance Liquid Chromatography (HPLC)^1,6	NA	HPLC¹⁵	NA	NA
Others	NA	Biochemical analysis (oxidative stress)¹⁸	TEWL measurements,¹⁵ HPLC (solubilisation of vitamin D3)¹⁵	NA	NA

NA = Not applicable; ELISA = enzyme-linked immunosorbent assay; PCNA = proliferating cell nuclear antigen; RT-qPCR = quantitative reverse transcription PCR; TEWL = transepidermal water loss; TTC = triphenyltetrazolium chloride; MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); MMPs = matrix metalloproteinases.

Disease pathology

The analytical techniques and methodology used in this research area varied according to the specific clinical condition being addressed. For histological analyses, haematoxylin and eosin (HE) staining was most commonly used to evaluate the overall morphology of the tissue after exposure to pathogens.^1,21,23 Special stains were also used — such as Fontana–Masson, for visualising melanin,¹⁶ Fite–Faraco/Ziehl–Neelsen to identify Mycobacterium leprae bacilli,²³ and Masson’s trichrome to explore changes in collagen.21 To assess the viability of the explants, colorimetric assays, such as 2,3,5-triphenyltetrazolium chloride (TTC)^1,6 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were used.²¹

To examine the expression of specific proteins, immunohistochemistry and immunofluorescence were employed — for example, when evaluating cell proliferation in psoriasis with labelled antibodies against the PCNA (Proliferating Cell Nuclear Antigen) marker and against specific immune cells that are characteristic of the condition.²¹ Western blotting and ELISA (Enzyme-Linked Immunosorbent Assay) were also used to measure the levels of specific proteins and cytokines involved in the inflammatory response, respectively.²¹ Among the articles analysed, gene expression data (obtained via real-time quantitative polymerase chain reaction (RT-qPCR)) further complemented the above molecular analyses, specifically in the context of: (i) visualising the immune response;^14,21,23 (ii) observing the interaction between the pathogen and the host;^14,23 and (iii) assessing the viability of the pathogen.^14,23 Reviewed studies that evaluated the skin permeation of compounds, specifically Dacarbazine¹ and Ibrutinib,⁶ both applied High-Performance Liquid Chromatography (HPLC) to quantify the tissue distribution of the compounds.

Cellular ageing

Saguie and collaborators¹⁸ included control groups of ex vivo skin tissue (DMEM without intervention) and UVA-exposed groups with the following exposure criteria: 16 J/cm² in total (either divided into 8 J/cm² on alternate days, or 4 J/cm² daily over 4 days) and a positive control of 150 J/cm² daily. Meanwhile, the experiments of Weihermann et al.²⁰ involved three types of cell/tissue model, namely: an ex vivo skin model; in vitro reconstituted artificial skin with keratinocytes and fibroblasts; and a coculture of fibroblasts and keratinocytes, which were all exposed to light ranging from 1 kJ/m² to 400 kJ/m². Both studies carried out morphological evaluations of the cells/tissues and employed immunohistochemistry to highlight damage to elastin after light exposure.^18,20 In the study by Saguie et al.,¹⁸ both biochemical analyses and Western blotting were performed, allowing, respectively, the visualisation of increased cellular oxidative stress and damage to the extracellular matrix (ECM), with the elevation of matrix metalloproteinases (MMPs). By using RT-qPCR, Weihermann et al. showed changes in elastin-related gene expression and elevated expression of MMPs.²⁰

Pharmacological formulation testing

Among the selected articles, experiments focused on evaluating tissue health and on specific cellular responses. Tissue health was evaluated by using a variety of analytical techniques, such as the MTT assay (for cell viability),¹⁷ HE staining (for assessing morphological aspects),¹⁷ and the measurement of transepidermal water loss (TEWL) to validate tissue integrity under the influence of chemical penetration enhancers (i.e. substances that facilitate the penetration of another chemical (in this case, vitamin D3) into the skin).¹⁵ To assess cell proliferation, direct counting and flow cytometry were commonly used, with a reduction in the number of fibroblasts and an increase in apoptosis after DMAE exposure.¹² In contrast, Romana-Souza and collaborators¹⁷ used immunohistochemistry to assess cell proliferation. Overall, protein quantification and gene expression analysis of cellular pathways (such as vasculogenesis, or lipid metabolism) after exposure to the test compounds — in this case, vitamin C¹⁷ and retinol¹³ — were performed by Western blotting¹⁷ and RT-qPCR,^13,17 respectively. With regard to skin-specific responses, HPLC was used to quantify the retention of vitamin D3 in specific skin layers and in the permeation fluid.¹⁵

Healing

The healing process was visualised through the monitoring of wound closure in the tissue sections by histopathological analysis.⁵ Regarding the assessment of cell proliferation, methodological differences were observed between the reviewed papers. In Pereira Oliveira et al.,⁵ immunohistochemistry was applied in situ to the ex vivo tissue, making it possible to determine cell proliferation via the Ki-67 marker, which was stimulated after exposure to nanoemulsions.⁵ Alencar-Silva et al.²² determined cell proliferation in the hOSEC model through the correlation of results obtained with in vitro assays on fibroblasts, in which similar gene expression was observed. This permitted further elucidation of the molecular mechanisms associated with proliferation and matrix remodelling that underlie the accelerated closure of an ex vivo lesion.²²

Cell culture optimisation

Overall, these studies sought to determine the feasibility of using ex vivo skin tissue-based models in different situations. Eberlin et al.¹⁹ indicated that the use of such models to test corrosion and skin irritation potential, based on the analysis of MTT assay data, was satisfactory, as the GHS (Globally Harmonized System of Classification and Labelling of Chemicals) classifications corresponded to the relevant OECD (Organisation for Economic Co-operation and Development) test guidelines (TG 431 and TG 439 for irreversible and reversible damage, respectively).¹⁹ On the other hand, for Frade and collaborators, the study focus was on the assessment of cellular health over a prolonged period of time in culture, through the detailed microscopic analysis of tissue morphology, as well as the immunostaining of specific molecular markers (Ck5/6, Ck10 and Ki-67).³

Discussion

This review analysed 16 Brazilian articles that utilised human skin collected from surgical procedures as a raw material for laboratory experiments. The findings highlighted that this practice remains limited due to poor methodological standardisation, which detrimentally affects the possibility of expanding the use of the technique to other applications, as well as to other geographical regions (i.e. there was a high concentration of research studies carried out within just a few Brazilian states). The reviewed studies originated mainly from the South-Eastern region of Brazil, and it was evident that most of the explants used were from plastic surgery procedures, mainly abdominoplasties. This may reflect the greater availability of plastic surgery facilities and their better links to research infrastructure in these regions.²⁴ It could also be coupled with a greater willingness to get involved with the donation of surplus tissue. This scenario, although favourable in terms of the extent of joined-up local expertise, also highlights the need to disseminate this technical and logistical know-how to other regions of the country.

Our analysis of the study objectives revealed the areas in which the explants are currently being used. These primarily involved the investigation of disease pathologies, cellular ageing and healing processes, as well as the pharmacological testing of cosmetics ingredients and the assessment of tissue culture protocols. These diverse applications and scientific reach could be promoted further throughout Brazil.

The heterogeneity of the methodologies employed, from obtaining the skin fragments to the tissue culture protocols used, highlighted the need for greater standardisation, with specific guidelines for each area of application. In addition, our review underlined the importance of initiatives to promote and disseminate information on the use of human skin-based models (which have high translational potential), by leveraging the plastic surgery procedures performed in the country.

Differences in tissue culture protocols

Culture maintenance period and frequency of media changes

The length of time that the ex vivo skin models were maintained in culture was identified as one of the major factors that differed between the study protocols. There appears to be a lack of consensus on the optimal duration of the experiments, as well as the frequency of culture medium changes. In the ‘disease pathology’ focus area, the models were used in short-term,^1,14,16 medium-term,^6,21 and long-term²³ experiments. This temporal variability reflected the importance of different intervals for each of the tissue responses, depending on the disease or condition being addressed. The culture medium renewal routine ranged from no changes (over a 4-day culture period)^14,¹⁶ to more than five (over a 60-day culture period).²³ Undertaking more than five culture medium changes over the extended (60-day) culture period employed by de Paula et al.,²³ resulted in greater microbiological control of the pathological model over the longer duration required, thus contributing to the successful study outcome.²³

Similar differences were also evident for the models used for the pharmacological testing of cosmetics ingredients. Experiments were either short-term (1 day in culture)^13,15 or medium-term (7–13 days in culture),^12,17 with culture medium exchanges ranging from zero to four in total, or more than five. Medium-term experiments (with between one and four medium changes) were also performed with the models, to investigate the areas of ‘cellular ageing’¹⁸ and ‘healing’.⁵ These medium-term experiments permitted, respectively, the induction of photoageing without compromising tissue integrity, as well as ensuring a continuous supply of regenerative factors for the healing process. In the study that sought to optimise cell culture conditions, the model demonstrated structural viability for up to 75 days, but did not specify the number of media changes, even though they were likely performed.³

This variability, in terms of medium changes and time in culture, reflects the adaptation of the protocols to suit different experimental purposes. However, it also highlights the need for minimum standard parameters that favour reproducibility and ease of transferability between laboratories.

Culture medium and format

Analysis of the protocols in the reviewed studies showed a preference for the use of DMEM as the tissue maintenance medium. However, its effectiveness is commonly enhanced by supplementation with 10% FBS, to recapitulate physiological conditions and optimise cell growth (see, for example, Eitan et al.²⁵). The frequent inclusion of antibiotics reflects concerns over preventing bacterial contamination, which can be a significant obstacle in primary tissue cultures. The addition of non-standard supplements, such as ʟ-glutamine, was evident; these are most likely required to fulfil the specific metabolic demands of a particular type of experiment. In most of the papers, the predominant type of culture setup was an air–liquid interface model,^{1,3,5,6,13–15,17–19,21–23} which is amenable to the development of a range of different experimental protocols. In this ex vivo culture maintenance format, a US-based group developed a technique aimed at improving the application of topical products, not only preventing contamination of the dermis by the product, but also reducing interaction between the epidermis and the culture medium.²⁶ This was achieved by optimising the use of the culture well insert to physically support the skin, so that the explant edges are able to form a better physical barrier.

The significance of these differences

The prevalence of short-term and medium-term studies in our analysis is directly aligned with the use of the ex vivo skin explants as models to mimic acute physiological conditions.^{1,5,6,12–19,21,22} The validity of this short/medium-term approach is supported by a lower loss of cell viability being evident over this particular timeframe, as well as a possible reduction in culture medium supplementation requirements. These factors were observed in a German study, which demonstrated the effective culture of full skin without the need for serum, that ensured viability for up to 72 hours (i.e. short-term).²⁷

One of the advantages of long-term studies is that they permit the observation of chronic processes, such as those in leprosy.²³ However, the limited number of longer-term studies in our analysis could be explained by the ongoing lack of more-robust models that are able to effectively maintain viability of the living tissues over a prolonged period. With this goal in mind, a Japanese group reported the use of microneedles for the administration of nutrient medium to the model, ensuring better oxygenation and greater tissue viability.²⁸

The adoption in Brazil of these types of innovative approaches, as proposed by research groups worldwide, would help Brazilian researchers to overcome some of the limitations identified in this review, such as the difficulty in maintaining tissue viability for long-term modelling.

Promoting the wider use of ex vivo human skin explant models in Brazilian research

From the papers selected from the literature search, it is clear that many areas of clinical relevance, such as severe burns treatment, chronic ageing and personalised medicine, are poorly represented in terms of the use of ex vivo human skin explant models in Brazil. Incorporating these human-based models within biomedical research would not only broaden the scope of their application, but also bring the basic research closer to clinical practice. For example, regarding severe burns treatment, a French study by Gross-Amat et al.²⁹ tested a topical composition for the treatment of second-degree burns. However, such severe burns were not covered in the reviewed studies by Brazilian-based researchers, which were focused on more-superficial burns — related to photoageing.^18,20

Internationally, human skin has been used for interesting and diverse research purposes. For example, one UK/US-based study used a system that keeps ex vivo human skin under optimised tension levels, in order to analyse the effects of different peels.³⁰ Furthermore, another international study highlighted the potential of human explants for the development of ‘personalised medicine’ approaches, in which multi-omic analyses of tissue are performed to individually evaluate molecular responses and optimise treatments, elucidating how individual genomic, transcriptomic, proteomic and metabolomic interactions occur during tissue repair.³¹ Another example of this approach was reported by a UK-based team, where an artificial intelligence model was developed and fed with multi-omic information related to the genome and transcriptome, as well as multimodal information.³² Preclinical tests with participants’ tissues were used as a basis to predict individual responses to drugs in patients with inflammatory bowel disease.³²

It is imperative to highlight here the significant absence of published scoping reviews that collate, analyse and summarise — in a manner analogous to the current study — the global literature on the use of ex vivo human skin explant models. The lack of such synthesised data makes it difficult to make direct comparisons, either between countries, or between Brazil and the rest of the world in general.

Current and future implications for Brazilian research

The use of hOSEC-based models shows huge potential for various basic and applied research applications. However, our review revealed a notable variability in tissue processing and maintenance methodologies, including the thickness of the skin fragment, the culture medium used, and the culture setup (i.e. air–liquid interface or submerged). This heterogeneity hinders standardisation of the protocols, which impacts on the results obtained and the method reproducibility between laboratories. Furthermore, the geographical distribution of studies is notably concentrated in the South-Eastern region of Brazil. This regional focus could, in effect, represent a barrier to the collaborative and diversified development and use of hOSEC-based models on a wider national scale. However, this barrier could be surmounted by the effective dissemination of standardised methodologies and guidelines throughout the country. With regard to these standardised methodologies and guidelines, some should be general, but others should be application-specific, to promote the widest possible uptake of the methods.

The formation of regional collaborative networks between laboratories with relevant expertise can investigate reproducibility in the short-term whilst, in the longer term, national networks can facilitate the expansion of technical and logistical know-how and promote the use of the methods within a wider range of research centres. Additionally, incentives through public calls for funding and specific calls for proposals for the use of hOSEC-based models would be effective strategies to stimulate the generation of new studies, expanding the use of the explant models into other areas, such as burns research.

Figure 2 summarises the current landscape of hOSEC model-based research in Brazil, illustrating the geographic concentration in the South-East of the country and the standard protocols employed. Furthermore, it highlights the transition from current uses to future applications, such as personalised medicine and burns studies, reinforcing the model’s potential as an ethical alternative to animal testing.

Figure 2.

A summary of the current landscape of hOSEC model-based research in Brazil.

Limitations of the review

The primary intrinsic limitation of this scoping review is the high level of heterogeneity apparent in the research protocols found in the literature. Many primary studies do not provide complete information and often exclude detailed information on, for example, the frequency of culture medium changes and other such critical parameters. This lack of methodological description prevents direct and rigorous comparisons between studies, hindering their reproducibility and potentially affecting the robustness of the data obtained.

A second limitation of our study concerns the exclusion of the so-called ‘grey literature’, such as conference abstracts, book chapters and unpublished or incomplete manuscripts. These items were not considered, and priority was given to full peer-reviewed published articles. We chose to restrict our analysis to this type of publication, because such articles typically provide more detailed and more rigorously evaluated information. Nevertheless, we acknowledge that grey literature may offer complementary insights, and we suggest that future reviews include and compare these sources to broaden the scope of the available evidence.

Conclusions

Our review of relevant Brazilian scientific research showed that ex vivo human skin explant models represent a versatile tool with great potential. However, their wider use could be hampered by the heterogeneity of the methods used, which makes it difficult to compare studies and can compromise their reproducibility. The creation and dissemination of standardised methodologies, as well as general and application-specific guidelines, are fundamental to progressing the wider use of the models. There is also the potential to expand the use of these models to other research areas that are currently under-represented, such as severe burns treatment. The wider use of these human-relevant models will contribute to the progressive reduction of animal use in experiments and consolidate their position as relevant and ethically preferable alternative models.

Supplemental material

Supplemental material - The use of skin explants in Brazil: A scoping review of applications, methodologies and research trends

Supplemental material for The use of skin explants in Brazil: A scoping review of applications, methodologies and research trends by Henrique Marchesim, Wilson Falco Neto, Bruna Cardoso Moscatel, Heloísa Coca Angélico, Livia Betteri Machiori, Pedro Henrique Soubhia Sanches, Eduardo Cavallini, Sara de Souza Costa and Ana Paula Girol in Alternatives to Laboratory Animals.

Footnotes

ORCID iD

Henrique Marchesim

Ethical considerations

All studies used will be appropriately cited and referenced, respecting copyright laws.

Funding

This study did not receive external funding.

Declaration of conflicting interests

The authors do not have any conflicts of interest associated with this study.

Supplemental material

Supplemental material for this article is available online.

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