Ex Vivo Human Skin as a Platform to Study Cosmetic Modulation of Specialized Pro-Resolving Mediators

Abstract

Chronic low-grade inflammation and oxidative stress induced by the exposome represent key drivers of skin aging and related imperfections. The development of experimental models suitable for studying these metabolic processes is therefore of primary importance for the cosmetic industry. In recent years, the role of specialized pro-resolving mediators (SPMs) in the resolution of inflammation has been highlighted; however, in vitro skin models to investigate them are still lacking. In this work, we developed an ex vivo human skin culture model that allows the quantification of maresin 1 (MaR1) production by measuring its concentration in the conditioned culture medium using an ELISA-based assay. The presence and survival of MaR1-synthesizing immune cells, namely Langerhans cells and leukocytes, were quantified during the first days of culture. The model’s ability to modulate MaR1 production was assessed in response to treatment with its precursor, docosahexaenoic acid (DHA), and with a DHA-rich cosmetic ingredient named Isochrysis Galbana Extract. Results demonstrated that the model produces MaR1 even in the absence of stimulation and responds to treatments with a further increase in MaR1 production. Furthermore, the tissue-to-medium ratio required to obtain MaR1 concentrations suitable for effective ELISA quantification was optimized. This model establishes a reproducible and scalable experimental platform for quantifying SPMs and evaluating DHA-based formulations, supporting both cosmetic research and mechanistic investigations.

1. Introduction

The exposome concept has provided a holistic framework for representing the wide range of environmental and lifestyle-related stressors to which individuals are exposed throughout their lives [1,2]. Aging, along with potential pathological disorders, arises from the complex interplay between the exposome and the genome [3,4], giving rise to distinct phenotypic changes that are unique to each individual. Low-grade chronic inflammation is one of the effects induced by the exposome through increased oxidative stress, which is recognized as a central driver of the aging process [5,6,7,8].

The skin is the organ most directly exposed to environmental stressors, including particularly harmful factors such as UV radiation, and it is also the one that most visibly manifests the effects of aging. The cosmetic industry has long been committed to developing products that enhance skin appearance, with the preservation of a youthful look as a primary goal. This objective is pursued not only by formulating treatments that improve key tissue parameters—such as hydration levels, stratum corneum composition, pigmentation, etc.—but also by studying the effects of biologically active ingredients on oxidative stress and the skin’s innate protective mechanisms against its damaging effects.

In the context of protective mechanisms against inflammation, lipid molecules involved in the physiological resolution of inflammatory processes—known as specialized pro-resolving mediators (SPMs)—have gained increasing attention in recent decades [9,10,11]. These compounds, which include lipoxins (Lx), resolvins (Rv), protectins (PD), and maresins (MaR), are primarily synthesized by immune cells from omega-3 polyunsaturated fatty acids (PUFAs) and act mainly on the same immune cells. Although SPMs are primarily known for their role in resolving inflammation, they also contribute to host defense, organ protection, and tissue remodeling [12,13]. The mechanism of action of SPMs in non-immune cells remains largely unknown; however, the expression of their receptors has been identified in various cell types. For instance, the ALX/FPR2 receptor, which binds LxA4 and RvD1, is expressed in both keratinocytes [13,14] and fibroblasts [15]. Additionally, DRV1/GPR32, which binds RvD1, and RORα/LGR6, which binds MaR1, are expressed in keratinocytes as well [14,16].

The cosmetic industry is therefore highly interested in identifying ingredients that can enhance the synthesis of SPMs and understanding their effects on skin biology. A simple and effective approach involves administering ingredients rich in SPM precursors, such as omega-3 PUFAs, which can be sourced from fish oils, microalgae extracts, or other plant-based materials [17,18,19,20,21,22,23]. To date, most dermatological research has focused on the treatment of chronic inflammatory skin conditions such as psoriasis and atopic dermatitis [9,18,24,25]. However, several cosmetic-grade applications aimed at promoting skin wellness have already been introduced. In this regard, a clinical trial showed that treatment with Anetholea anisata extract enhanced the synthesis of LxB4, RvD1, and RvD2, leading to beneficial effects on scalp barrier restoration and improvement of dandruff conditions [10]. Interestingly, Pagac et al. [26] demonstrated that the scalp microbiome, particularly Malassezia yeasts, can metabolize omega-3 PUFAs into oxylipins, including SPMs, which exert beneficial effects.

Regarding sun protection cosmetics, Martinez et al. [27] showed that ligands targeting ALX/FPR2, the receptor for LxA4 and RvD1, can mitigate UVB-induced skin damage in hairless mice, including epidermal thickening, sunburn cell formation, and collagen degradation. Similarly, protective effects on skin homeostasis against UVB-induced inflammation have also been observed for MaR1, which can counteract the upregulation of multiple inflammatory markers while preserving the cellular antioxidant defense system [28]. Additional studies in mice confirm similar protective effects linked to the production of RvD1 and RvD5 [29,30].

However, research in this particular field remains limited, and the development of experimental models specifically designed to investigate SPM metabolism in human skin is a crucial factor. In general, the existing literature on this topic relies on in vivo experiments or 3D skin models. Inflammatory ex vivo skin models characterized for their immune cell composition are still scarce [31,32,33,34], and their application for SPM characterization remains highly limited. Additionally, most studies on the ex vivo skin immune system have focused on characterizing Langerhans cells (LCs), which are located in the epidermis. Rakita et al. [35] have thoroughly documented the presence of LC and T cells in a wound model based on the suction blistering procedure; however, the behavior and fate of these cells during the ex vivo culture remain unclear.

Furthermore, in studies based on organotypic skin cultures, SPM characterization has typically been performed by treating the tissue with solid-phase extraction, followed by chromatographic analysis of the extracts. These procedures are labor-intensive and not well-suited for routine high-throughput screenings.

In this study, we aimed to develop an ex vivo skin culture model to assess the modulation of SPM synthesis (specifically MaR1), without relying on solid-phase extraction. This model was designed to evaluate the skin’s response to the topical application of omega-3 PUFA precursors, particularly docosahexaenoic acid (DHA).

To achieve this, we carried out the following experimental steps:

• Since immune cells (primarily neutrophils and macrophages) are the main producers of SPMs, we estimated the LCs in the epidermis and leukocytes in the dermis to assess the model’s ability to generate detectable amounts of SPMs.
• The survival and retention of these cells during the first 3 days of culture were studied to rule out potential adaptation issues or migration out of the tissue.
• It was demonstrated that MaR1 is produced at detectable levels and released into the culture medium, where it was quantified using an ELISA assay.
• The findings confirmed that the model can effectively modulate MaR1 production in response to DHA treatment.

Overall, ELISA analysis of conditioned medium from ex vivo skin cultures has proven to be an effective approach for studying SPMs metabolism, with MaR1 serving as a representative biomarker.

2. Materials and Methods

2.1. Ex Vivo Skin Culture

The skin samples were collected as discarded tissue from plastic surgery procedures with no subsequent intended use, after obtaining the donors’ informed consent. Once excised, skin samples were placed in plastic containers containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL) and maintained at 4 °C during transport until their arrival at the laboratory, where they were processed according to the procedures established for Cutech’s proprietary ex vivo skin model. Skin tissues intended for culture were processed within 24 h of collection by carefully removing the hypodermis and then excising cylindrical samples (8 mm in diameter) using an electronic punch device (Nouvag AG, mod. TCM3000BL, Goldach Switzerland). The obtained biopsies were cultured inserted in sterilized medical stainless-steel rings, with the epidermal portion exposed to air and the dermal portion immersed in a modified William’s E culture medium. The cultures were maintained under standard cell culture conditions (37 °C, in a 5% CO₂/95% air-humidified incubator), in six-well plates at a density of two to four biopsies per well with 2.0 or 2.5 mL of medium, depending on the biopsies thickness.

Table 1. Donor characteristics and sample information. The skin phototype is expressed according to the scale by Del Bino et al. [36].

N.	Sex	Age	Phototype (ITA°)	Body Region	Analysis Performed
1	Female	36	48—Light	Abdomen	Immune cells estimation
2	Female	70	49—Light	Abdomen	Immune cells estimation
3	Female	48	22—Tanned	Abdomen	Immune cells estimation
4	Female	43	39—Intermediate	Abdomen	Immune cells estimation
5	Male	71	39—Intermediate	Abdomen	Immune cells estimation
6	Male	54	26—Tanned	Abdomen	MaR1 quantification
7	Female	37	39—Intermediate	Abdomen	MaR1 quantification
8	Female	55	46—Light	Abdomen	MaR1 quantification
9	Male	48	48—Light	Abdomen	MaR1 quantification
10	Female	59	30—Intermediate	Breast	MaR1 quantification
11	Female	61	47—Light	Abdomen	MaR1 quantification
12	Female	50	25—Tanned	Abdomen	MaR1 quantification
13	Female	42	38—Intermediate	Breast	MaR1 quantification
14	Female	56	19—Tanned	Abdomen	MaR1 quantification

summarizes the available demographic information of the donors from whom the skin samples were obtained. Since samples were provided with only the donor’s year of birth, the age was estimated as the difference between the year of collection and the reported year of birth. Skin phototype was determined using a spectrocolorimeter (SkinColorCatch™, Delfin Technologies, Kuopio, Finland) and classified according to Del Bino et al. [36]. Because the primary objective of this study was to establish a reliable method for evaluating SPM production, and not to analyze donor-related variability, these demographic data are presented for descriptive purposes only.

2.2. Methodological Approach for Immune Cell Quantification in Ex Vivo Skin Samples

To verify whether the ex vivo skin model was suitable for studying SPM metabolism, histological and immunohistochemical analyses were performed targeting the following markers:

• CD1α, a marker specifically expressed in LCs, the most abundant immune cell population within the epidermal layer [37], and
• CD18, also known as integrin β chain-2 (β2), an integrin β chain protein shared among all leukocytes. It pairs with one of several variants of α chains (CD11), depending on the immune cell subtype, forming various heterodimers.

Regarding CD11, the known variants CD11a, CD11b, CD11c, and CD11d, are expressed on different immune cell types, giving rise to major integrins such as LFA-1 (Lymphocyte function-associated antigen 1, CD11a/CD18), αMβ2 (Mac-1, CD11b/CD18) and αDβ2 (CD11d/CD18) [38]. These heterodimers play critical roles in cellular adhesion, cell surface signaling, and immune regulation. Since CD18 is the common subunit shared across integrins expressed by diverse immune cells, it was chosen as a broad marker to identify the entire leucocyte population within the skin.

Immunohistochemical staining targeting the aforementioned markers was performed upon sample arrival at the laboratory, and subsequently, in different donors, at 24, 48, and 72 h of culture. At the time of harvesting, skin biopsies were fixed in 4% formaldehyde, paraffin-embedded and sectioned into 5 μm-thick slices using a Leica RM 2255 microtome. The sections were processed for immunostaining with the BOND-III automated stainer (Leica Biosystems, Nussloch, Germany) with anti-CD1α (Ab233990, Abcam, Cambridge UK) or anti-CD18 (Abcam, Ab307406) antibodies, and the DS9390 detection system (Leica Biosystems). Sections were mounted with Fluoromount aqueous mounting medium (cat. F6057, Merck KGaA, Darmstadt, Germany) containing DAPI for nuclear counterstaining, and digital images in fluorescence were captured using a Leica DFC9000GT digital camera mounted on a Thunder Imager (Leica Biosystems).

The absence of non-specific antibody binding was verified during the protocol optimization phase by including negative controls in which the primary antibody was omitted. No detectable background signal was observed under these conditions. For image acquisition, fields were selected while excluding approximately 15% of the tissue area near the biopsy edges and regions exhibiting structural irregularities (e.g., convolutions, hair follicles, or large vessels). Field selection was performed under blue fluorescence (DAPI) illumination to avoid visualizing immune cell distribution and thereby minimize observer bias. Images were analyzed using ImageJ application (NIH, USA). The number of CD1α⁺ cells (LCs) was expressed as percentage of all nucleated cells in the epidermis, whereas CD18⁺ cells were expressed in number of positive cells per mm² of dermis area.

2.3. Experimental Design and Estimation of MaR1 Production in Ex Vivo Skin Cultures

The initial experiments aimed to evaluate the suitability of ex vivo skin cultures as a preclinical model for studying SPM metabolism. Specifically, the experiments were designed to evaluate the ability of ex vivo tissue to produce MaR1 at levels sufficient for its release into the culture medium at concentrations measurable by ELISA, and to modulate its production in response to topical treatments.

MaR1 levels were quantified using a commercial ELISA kit (Cat. No. 501152, Cayman Chemical, Ann Arbor, MI, USA), based on a competitive binding assay in which sample-derived MaR1 competes with MaR1 conjugated to acetylcholinesterase for a limited number of MaR1-specific rabbit antiserum binding sites. According to the manufacturer’s specifications, the assay displays cross-reactivity below 0.01% with DHA (the polyunsaturated fatty acid precursor from which MaR1 is synthesized) and with SPMs other than MaR1. All technical control groups required by the ELISA plate, as well as the standard curve, were prepared by diluting the standards in the same medium used for the skin cultures, to compensate for any potential interfering factors in MaR1 quantification. The ELISA plate also included control wells containing unconditioned culture medium, used as the maximum binding reference. Only sample data falling within 20–80% of the optical density measured in this maximum binding control were considered reliable and included in the quantitative analysis.

The total amount of MaR1 released into the culture medium was calculated by multiplying the measured concentration by the volume of conditioned medium. Following preliminary experiments performed to evaluate inter-donor variability in MaR1 release, MaR1 production was normalized to the biopsy biomass to determine the release ratio for each experimental group. More in detail, a preliminary experiment was conducted to evaluate whether the biopsy-to-medium ratio was sufficient to achieve detectable MaR1 concentrations.

2.3.1. Effect of Tissue-to-Medium Ratio and Culture Time: Preliminary Study

In the absence of any information regarding MaR1 production and its stability over time, a preliminary experiment was designed to determine whether it would be more effective to increase the biopsy-to-medium ratio over a shorter culture period or to maintain standard culture conditions for a longer duration. For this purpose, two experimental groups were compared: one group consisting of four biopsies cultured in 2.5 mL of medium for 24 h, and a second group with two biopsies cultured in 2.5 mL of medium for 48 h. Based on the results obtained, it was decided to proceed with the standard culture conditions, i.e., two biopsies in 2.5 mL of medium, and to collect the conditioned medium after 48 h (day 2 of culture).

2.3.2. Response to Stimulatory Treatments and Final Protocol with Optimized Biopsy-to-Medium Ratio

In order to evaluate the ability of the ex vivo model to modulate MaR1 synthesis in response to specific treatments, selected groups were treated with DHA dissolved in ethanol, to assess its effect compared to controls receiving the same volume of ethanol. Based on the results obtained, a revised screening protocol was established, relying on an optimized biopsy-to-medium ratio, designed to achieve detectable MaR1 concentrations (i.e., within the ELISA sensitivity range of approximately 9.6–186.1 pg/mL) even using biopsies collected from donors characterized by a low endogenous production of this SPM. In the conclusive experiments, some groups were included to evaluate the effect of a microalgal extract containing 7% DHA, obtained by CO₂ extraction and listed under the International Nomenclature of Cosmetic Ingredients (INCI) as Isochrysis galbana Extract (hereinafter abbreviated as IGE). All topical treatments were administered dissolved in 2 µL of ethanol per biopsy, and the same vehicle was also administered to the control group.

2.4. Methodological Considerations and Data Limitations

The data analysis was primarily aimed at characterizing the functional performance of the ex vivo skin model and establishing a method for assessing the presence and modulation of SPMs, with MaR1 chosen as a representative molecule. Donor information is reported for descriptive purposes only and was not intended to represent population-related variability or age-dependent differences. ELISA results were generally obtained by averaging two technical replicates within the same plate, except in a few cases where three replicates were performed; therefore, only descriptive statistics were applied.

Regarding the quantification of immune cells, it should be noted that counting labeled cells in histological sections can be subject to considerable inaccuracy and may be influenced by the observer, due to signals originating from cells located in a different plane than the section under analysis and other technical problems [39,40,41,42]. In this context, our data should be considered as indicative.

3. Results

3.1. Quantification of the Immune Cells in Ex Vivo Skin Samples

The immunohistochemical staining confirmed a substantial presence of immune cells in the skin samples, with LCs concentrated in the epidermis, while leukocytes were predominantly located in dermal areas surrounding small blood vessels (Figure 1).

Table 2. Abundance of CD1α⁺ and CD18⁺ cells in histological sections (n, number of cells) from skin samples collected from the abdomen of four donors upon arrival at the laboratory. Data are expressed as the percentage of total nucleated epidermal cells (i.e., CD1α⁺ + other cells) for CD1α⁺ cells, and as the number of cells per mm² of dermis for CD18⁺ cells. Mean values are based on cell counts from images of three microscope fields per donor. In the last columns, the mean and corresponding standard deviation are calculated from the pooled data (twelve histological fields).

	Donor 1		Donor 2		Donor 3		Donor 4		Pooled Data
Variable	Mean	Std. Dev.	Mean	Std. Dev.	Mean	Std. Dev.	Mean	Std. Dev.	Mean	Std. Dev.
CD1α+ cells (n)	8.33	2.08	6.33	0.58	6.67	2.08	6.67	3.06	7.00	2.00
Other cells (n)	253.00	39.69	213.33	19.22	262.67	62.69	253.00	47.44	245.50	43.25
CD1α+/total cells (%)	3.30	1.15	2.90	0.37	2.45	0.27	2.49	0.68	2.78	0.70
CD18 cells (n)	27.0	4.36	37.7	19.6	61.7	21.2	81.3	7.5	51.92	25.54
Area (mm2)	0.095	0.01	0.30	0.01	0.32	0.01	0.34	0.02	0.26	0.10
CD18+ cells/mm2	285.21	39.43	128.4	71.4	194.5	76.2	240.5	16.4	212.31	77.55

shows the quantification data of immune cells, specifically LCs (CD1α⁺) in the epidermis and leukocytes (CD18⁺) in the dermis, estimated from abdominal skin samples collected from four donors. By pooling the data obtained from all donors (12 images), the estimated abundance of LCs was 2.8% of epidermal cells, while the dermal sections contained approximately 212 CD18⁺ cells per mm². Following these preliminary results, a three-day experiment was conducted to evaluate changes in the abundance of CD1α⁺ and CD18⁺ cells by analyzing twelve section images for each day of culture. The results (

Table 3. Abundance of CD1α⁺ and CD18⁺ cells (n, number of cells) in histological sections from skin samples collected from donor 5, and their variation over the culture period. Cell abundances are expressed as the percentage of total nucleated epidermal cells (i.e., CD1α⁺ + other cells) for CD1α⁺ cells, and as the number of cells per mm² of dermis for CD18⁺ cells. Each mean value is based on cell counts from twelve histological sections.

Culture Time	Epidermis				Dermis
	Variable	Mean	Std. Dev.	Δ vs. Day 0 (%)	Variable	Mean	Std. Dev.	Δ vs. Day 0 (%)
Day 0	CD1α+ cells (n)	10.1	2.6		CD18+ cells (n)	35.3	8.5
	Other cells (n)	271.6	30.9		Area (mm2)	0.2	0.0
	% CD1α+ cells	3.6	1.0	0	CD18+ cells/mm2	170.8	45.0	0
Day 1	CD1α+ cells (n)	8.4	1.6		CD18+ cells (n)	34.2	14.6
	Other cells (n)	293.5	78.6		Area (mm2)	17.0	58.3
	% CD1α+ cells	2.9	0.7	−19.5	CD18+ cells/mm2	164.4	87.5	−3.7
Day 2	CD1α+ cells (n)	7.3	2.1		CD18+ cells (n)	21.3	8.2
	Other cells (n)	293.6	59.0		Area (mm2)	0.2	0.0
	% CD1α+ cells	2.5	0.6	−31.6	CD18+ cells/mm2	107.3	42.8	−37.2
Day 3	CD1α+ cells (n)	4.5	1.7		CD18+ cells (n)	19.5	11.8
	Other cells (n)	278.8	48.0		Area (mm2)	34.5	80.1
	% CD1α+	1.6	0.5	−55.8	CD18+ cells/mm2	84.2	65.9	−50.7

) broadly confirm the previous findings, showing that a substantial decline in cell abundance occurs between day 1 and day 2 of culture, with a loss of approximately 50–60% of cells by day 3.

Taken together, the findings highlight the marked variability in immune cell abundance among different donors, although the values remained comparable in order of magnitude. Importantly, the changes in cell abundance observed during culture could also differ substantially between donors.

3.2. Estimation of MaR1 Synthesis in Ex Vivo Skin Cultures

The results of a preliminary experiment, summarized in

Table 4. Quantification of MaR1 released by four biopsies cultured for 24 h versus two biopsies cultured for 48 h (donor n. 6), and estimation of the release normalized to the wet weight (w.w.) of skin. Three ELISA technical replicates (t.r.) were performed for each sample.

Experimental Group	Medium (mL)	Culture Duration (Days)	t.r.	MaR1 Conc. (pg/mL)	Std. Dev. (pg/mL)	Skin w.w. (g)	MaR1 Normalized on the Skin w.w. (pg/g)
4 biopsies	2.5	1	3	137.05	10.03	0.699	490.15
2 biopsies	2.5	2	3	48.86	5.15	0.307	398.14

, confirm the ability of the ex vivo human skin model to synthesize MaR1 and release it into the culture medium at concentrations detectable by ELISA. The amount of MaR1 released by four biopsies after 24 h was more than twice that released by two biopsies after 48 h. When normalized to skin wet weight, the data further confirm that the 4-biopsies group cultured for 24 h produced higher levels of SPM compared to the 2-biopsies group cultured for 48 h, although normalization reduces the difference to less than 25%. This finding suggests that MaR1 release does not occur at a constant rate but is mainly concentrated during the initial adaptation phase following biopsy excision. An alternative explanation is that MaR1 may partially degrade during the longer culture period. Following the preliminary experiment, although higher MaR1 production was observed with four biopsies per well, the culture protocol was initially standardized to two biopsies cultivated for 48 h. This condition was chosen because MaR1 reached measurable concentrations while providing a longer culture period suitable for testing experimental treatments.

In the following experiments (

Table 5. Concentration values of MaR1 measured by ELISA in the conditioned medium after 2 days of ex vivo skin culture. Each biopsy received 2 µL of ethanol on day 0 and again on day 1, either in its pure form (control) or containing DHA or IGE in the specified amounts. When multiple dilutions of the conditioned medium with fresh culture medium were tested, only the result for the lowest dilution within the assay’s sensitivity range is reported. Two ELISA technical replicates were performed for each sample. Abbreviations: t.r., technical replicates; d.f., dilution factor.

Donor	Group		MaR1 (pg/mL)
		t.r.	Mean	Std. Dev
N. 7	Control	2	25.98	4.01
	DHA 0.5 mg/biopsy (d.f. 1:70)	2	10,245.78	352.65
N. 8	Control	2	10.37	0.16
	DHA 0.5 mg/biopsy (d.f. 1:50)	2	2161.97	137.29
	DHA 0.07 µg/biopsy	2	59.03	0.48
	IGE 1 µg/biopsy	1	24.76	single value
	IGE 0.5 µg/biopsy	2	18.81	0.50

), MaR1 production in skin samples treated only with ethanol (controls) was compared with samples treated with DHA at 0.5 mg/biopsy (positive control) and with IGE at two concentrations. Additionally, a control group treated with DHA at 0.07 µg/biopsy, corresponding to the DHA content present in the IGE treatment at 1 µg/biopsy, was included. During the ELISA assay, the conditioned media from the positive control group were initially diluted between 1:35 and 1:140 with fresh culture medium, and subsequent experiment between 1:50 and 1:100, whereas all other experimental groups were analyzed either undiluted or at dilutions up to 1:10.

In both the experiments, the skin samples responded to treatment with 0.5 mg/biopsy DHA by markedly increasing MaR1 release, by approximatively 200- to 400-fold, in a strongly donor-dependent manner. Focusing on the second experiment, administration of DHA at 0.07 µg/biopsy resulted in an approximately 5- to 6-fold increase in MaR1 levels compared to the control group. Treatment with IGE resulted in an approximate 1.9–2.5 folds increase in MaR1 levels compared to the control group.

Based on these results, a series of additional experiments was carried out to assess the variability in MaR1 production among different donors. These experiments included a treatment with 0.5 mg/biopsy of DHA as a positive control, along with various concentrations of DHA or IGE as stimulatory treatments at doses compatible with cosmetic product formulations. The results are presented in

Table 6. MaR1 release from different donors, quantified after 2 days of culture (including data from Table 4) and normalized to the wet weight (w.w.) of the skin samples. When multiple dilutions of the conditioned medium (d.f., dilution factor) were tested, only the lowest dilution within the assay’s sensitivity range is reported, or, when all concentrations were below the detection limit (B.D.L.), the lowest dilution tested. Two or three ELISA technical replicates (t.r.) were performed per sample.

Donor	Experimental Group	Medium (mL)	t.r.	MaR1 (pg/mL)	Std. Dev. (pg/mL)	Skin w.w. (g)	MaR1 Normalized on Skin w.w. (pg/g)
N. 9	Control (d.f. 1:2)	2.5	2	B.D.L.		0.3239	Unquantifiable
	DHA 0.5 mg/biopsy (d.f. 1:50)	2.5	2	4519.41	351.65	0.3491	32,364.72
N. 7	Control	2.5	2	25.98	4.01	0.2878	225.71
	DHA 0.5 mg/biopsy (d.f. 1:50)	2.5	2	10,245.78	352.65	0.3064	83,598.07
N. 10	Control	2.5	2	15.80	0	0.3142	125.75
	DHA 0.5 mg/biopsy (d.f. 1:50)	2.5	2	3211.16	397.60	0.3161	25,396.70
	DHA 0.75 µg/biopsy	2.5	2	36.58	0.72	0.2835	322.60
	DHA 0.072 µg/biopsy	2.5	2	23.26	2.25	0.3065	189.72
N. 8	Control	2.5	2	10.37	0.16	0.2627	98.67
	DHA 0.5mg/biopsy (d.f. 1:50)	2.5	2	2161.96	137.29	0.2707	19,966.40
	DHA 0.072 µg/biopsies	2.5	2	59.03	0.48	0.2735	539.61
	IGE 1 µg/biopsy	2.5	1	24.76	-	0.2302	268.87
	IGE 0.5 µg/biopsy	2.5	2	18.81	0.50	0.24	195.96
N. 11	Control	2.5	2	B.D.L.		0.2273	Unquantifiable
	DHA 0.5 mg/biopsy (d.f. 1:50)	2.5	2	815.84	288.57	0.2603	7835.56
	DHA 0.14 µg/biopsy	2.5	2	B.D.L.		0.2339	Unquantifiable
	DHA 0.028 µg/biopsy	2.5	2	B.D.L.		0.2634	Unquantifiable
	DHA 0.0056 µg/biopsy	2.5	2	16.57	0.81	0.1968	210.46
	IGE 2 µg/biopsy	2.5	2	20.22	4.17	0.285	177.34
	IGE 0.4 µg/biopsy	2.5	2	B.D.L.		0.2665	Unquantifiable
	IGE 0.08 µg/biopsy	2.5	2	B.D.L.		0.2139	Unquantifiable
N. 12	Control	2	3	B.D.L.		0.1931	Unquantifiable
	DHA 0.5 mg/biopsy (d.f. 1:50)	2	3	B.D.L.		0.1881	Unquantifiable
	DHA 0.7 µg/biopsy	2	3	B.D.L.		0.2257	Unquantifiable
	DHA 0.14 µg/biopsy	2	3	B.D.L.		0.2131	Unquantifiable
	DHA 0.028 µg/biopsy	2	3	B.D.L.		0.2219	Unquantifiable
	DHA 0.0056 µg/biopsy	2	3	B.D.L.		0.2223	Unquantifiable
	IGE 10 µg/biopsy	2	3	38.42	3.36	0.1899	404.64
	IGE 2 µg/biopsy	2	3	16.04	5.45	0.2198	145.96
	IGE 0.4 µg/biopsy	2	3	B.D.L.		0.2211	Unquantifiable
	IGE 0.08 µg/biopsy	2	3	B.D.L.		0.2729	Unquantifiable

, where the total amount of MaR1 released into the medium was normalized to the total fresh biomass of the skin biopsies.

The data indicate that different donors exhibited substantial variability in their capacity to synthesize MaR1. In many cases, both the control group and some experimental treatments yielded values below the working range of the ELISA used.

Considering the overall results from Table 4, Table 5 and Table 6, after 2 days of culture the amount of MaR1 released by the control group—when within the assay’s measurable range—ranged from 99 to 398 pg/g w.w. of skin. However, many donors showed a lower production capacity, releasing MaR1 at concentrations below the working range of the ELISA assay.

These findings indicate that, for each experimental condition, approximately 400 mg of skin cultured in 2.5 mL of medium are required to obtain conditioned media with MaR1 concentrations within the sensitivity range of the adopted ELISA. The number of biopsies (8 mm diameter in the Cutech ex vivo skin model) required to achieve this biomass-to-medium ratio depends on the skin sample thickness and may range from 2 to 4 biopsies.

Table 7. Quantification of MaR1 release after 2 days of culture, normalized to the wet weight (w.w.) of skin samples. Only the conditioned medium from the positive control group (DHA, 0.5 mg/biopsy) was diluted with fresh medium at ratios ranging from 1:35 to 1:200, depending on the experiment; the result corresponding to the lowest dilution within the assay’s sensitivity range is reported. Three biopsies per well were seeded in the first experiment, while four biopsies per well were used in the second. Two ELISA technical replicates (t.r.) were performed for each sample.

Donor	Experimental Group	Medium (mL)	Dil. Factor	t.r.	MaR1 (pg/mL)	Std. Dev. (pg/mL)	Skin w.w. (g)	MaR1 (pg/g Skin)	Variation vs. Control (%)
N. 13	Control	2.5	1	2	33.30	0.51	0.4271	194.90	0.00
	DHA 0.5mg/biopsy	2.5	140	2	23,942.27	472.90	0.4650	128,721.89	65,945.93
	DHA 0.7 µg/biopsy	2.5	1	2	51.49	10.14	0.4117	312.65	60.42
	DHA 0.14 µg/biopsy	2.5	1	2	29.90	2.83	0.4042	184.94	−5.11
	DHA 0.028 µg/biopsy	2.5	1	2	36.60	0.53	0.3444	265.64	36.30
	IGE 10 µg/biopsy	2.5	1	2	44.95	3.13	0.4647	241.81	24.07
	IGE 2 µg/biopsy	2.5	1	2	27.54	1.35	0.3436	200.36	2.80
	IGE 0.4 µg/biopsy	2.5	1	2	20.45	1.37	0.4191	122.00	−37.40
N. 14	Control	2.5	1	2	35.31	1.53	0.4650	189.84	0.00
	DHA 0.5mg/biopsy	2.5	200	2	25,853.00	1871.05	0.5151	125,475.62	65,995.27
	DHA 0.7 µg/biopsy	2.5	1	2	73.86	2.66	0.4706	392.37	106.68
	DHA 0.14 µg/biopsy	2.5	1	2	38.81	2.65	0.4711	205.95	8.49
	DHA 0.028 µg/biopsy	2.5	1	2	42.50	2.59	0.4467	237.87	25.30
	IGE 10 µg/biopsy	2.5	1	2	100.35	6.25	0.4416	568.12	199.26
	IGE 2 µg/biopsy	2.5	1	2	44.12	4.24	0.4879	226.06	19.08
	IGE 0.4 µg/biopsy	2.5	1	2	25.89	1.21	0.5078	127.47	−32.86

presents the results of two experiments in which skin biopsies, with a total group biomass ranging from 344 to 515 mg, were seeded and cultured in 2.5 mL of medium. In the first experiment, this required 3 biopsies per well, while in the second, 4 biopsies per well were used.

The improved experimental protocol allowed the detection of measurable MaR1 concentrations in all groups. When normalized to the weight of the skin biopsies, concentrations were very similar between the control groups in both experiments, as well as between the positive controls (DHA 0.5 mg/biopsy), which showed MaR1 release approximately 660 times higher than the controls. These two donors responded positively to treatment with DHA at 0.7 µg/biopsy and the corresponding IGE treatment at 10 µg/biopsy, increasing the MaR1 production by 60–100% and 24–200%, respectively.

In terms of percent change versus control, these donors exhibited lower responsiveness to certain stimulatory treatments compared to others reported in Table 6, where MaR1 production increased substantially even after treatment with lower doses of DHA or IGE. Therefore, donor responsiveness to stimulatory treatments might be related to the baseline level of MaR1 production detected in the control group. In some donors, the control group showed lower MaR1 values of approximatively 100 pg/g of skin w.w., or possibly even lower in experiments where MaR1 was below the detection limit. Based on the present data, lower baseline MaR1 production appears to be associated with greater responsiveness to mild stimulatory treatments.

4. Discussion

Ex vivo skin culture is the experimental model that most closely retains the anatomical organization of in vivo skin, although some functional alterations occur during culture [33,43,44]. Previous studies on the skin immune cells have primarily focused on LCs and their function as professional antigen-presenting cells [45,46]. The abundance of LCs has been reported to range from approximately 2% to 3–5% of the total epidermal cell population [39,47,48], and their functional stability in culture has been shown to persist for at least three days [45]. Our findings based on samples from four donors indicate that LCs can range between 2.4% and 3.3% of epidermal cells, and tend to decline by approximately 56% after three days of culture. Although it has not been conclusively demonstrated that LCs contribute to the production of SPMs, there is strong reason to believe they may play a role. In line with that, 15-LOX-1, a key enzyme in the biosynthetic pathway of SPMs [49,50,51], is highly expressed in LCs of individuals with contact dermatitis and atopic dermatitis [52]. Moreover, LCs are ontogenetically related to macrophages [53], which, together with neutrophils, are among the primary producers of SPMs [12]. Therefore, LCs possess both the enzymatic machinery and the cell lineage derivation consistent with the ability to synthesize SPMs.

Even more relevant was the presence of leukocytes, i.e., the primary producers of SPMs in the dermis, with an estimated density ranging approximately from 128 to 285 cells per mm² (Table 2), which declined by about 50% after three days of culture (Table 3). Although the quantification of leukocyte abundance in healthy skin remains relatively underexplored, these findings are consistent with previous reports in terms of order of magnitude [54,55].

These data supported the hypothesis that ex vivo skin retains a sufficient number of competent cells for SPM synthesis. A case study on SPM modulation in ex vivo skin explants was reported by Amandine et al. [56], using solid–liquid extraction followed by LC–MS/MS quantification. While this technique represents the gold standard for SPM quantification [57], it is not suitable for high-throughput screening, unlike the ELISA assay. Interestingly, in the cited study, no terminal DHA- or EPA-derived SPMs were detected, despite the presence of their intermediate precursors among the lipidomic metabolites found in the skin extract. These results are in line with the findings reported by Kendall et al. [58], who applied comparable methodological approaches. Therefore, to the best of our knowledge, the present study represents the first documented case of MaR1 production in ex vivo skin culture. Furthermore, prior to this work, no conclusive data were available to determine whether such production could reach concentrations above the ELISA detection threshold in the conditioned medium.

The results demonstrated that even untreated skin (control group) was capable of producing measurable amounts of MaR1, provided that the tissue-to-medium weight ratio exceeded a defined threshold. In the Cutech ex vivo skin model, this threshold corresponded to approximately 160 mg of w.w. skin/mL. It is plausible that the amount of MaR1 produced in the absence of any treatment was influenced by tissue stress in response to biopsy excision and adaptation to culture conditions, and may also be affected by the donor’s physiological condition at the time of excision. Furthermore, the leaching of SPMs into the culture medium at concentrations compatible with ELISA quantification could be limited by their lipid nature, due to low solubility. Furthermore, important limitations to the quantitative comparability and reproducibility between studies arise due to donor heterogeneity, short culture time, and absence of systemic circulation.

It is worth noting that MaR1 originates from DHA, which, in absence of dietary intake, is synthesized with very low efficiency from alpha-linolenic acid (18:3n-3) [59,60]. In our experiments, treatment with DHA at 0.5 mg/biopsy led to a 200 to 660-fold increase in MaR1 concentration in the medium, supporting its use as a positive control in our experimental setup. This result confirms that immune cells retained their functionality and stimulus-responsiveness during early culture, demonstrating that the ex vivo model is suitable for studying the metabolism of SPMs.

Treatment with IGE, a microalgae-derived cosmetic ingredient, administered at concentrations consistent with cosmetic formulations (1–10 µg per biopsy, equivalent to 0.02–0.2 µg/mm², delivering approximately 0.07–0.7 µg of DHA per biopsy), resulted in an increase in MaR1 concentration in the culture medium. The minimal effective dose of both DHA and IGE required to modulate MaR1 production showed considerable variability depending on donor responsiveness.