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Article

Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT)

by
Yuka Sawada
1,
Hanae Tsukumo
2,
Junji Fukuda
3,
Kazutoshi Iijima
3,* and
Hiroshi Itagaki
3,4
1
Department of Chemistry and Life Science, Graduate School of Engineering Science, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
2
Department of Chemical and Energy Engineering, Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
3
Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
4
Itagaki Cosmetics Safety Consulting, 5-11-19-2504 Minamidai, Minami-ku, Sagamihara 252-0314, Japan
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(12), 6207; https://doi.org/10.3390/app12126207
Submission received: 20 April 2022 / Revised: 9 June 2022 / Accepted: 15 June 2022 / Published: 18 June 2022
(This article belongs to the Section Applied Biosciences and Bioengineering)
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Abstract

:
To improve the accuracy of skin sensitization prediction of chemicals by conventional alternative methods using cells, it is important to reproduce the environment of skin in vitro, such as the crosstalk between keratinocytes and dendritic cells (DCs). We developed a skin sensitization test system based on the markers and criteria of the human cell line activation test (h-CLAT), which combines THP-1 cells as a surrogate for DCs and keratinized normal human epidermal keratinocytes (NHEK). After exposure to chemicals via keratinized NHEK, the cell surface expression of CD54 and CD86 on THP-1 was measured by flow cytometry. This co-culture system evaluated 2,4-dinitrochlorobenzene (DNCB), a typical sensitizer, as positive, lactic acid (LA), a non-sensitizer, as negative, and isoeugenol (IE), a prohapten that requires biological activation to acquire skin sensitization, as positive. However, the expression levels of CD54 and CD86 in DNCB-treated THP-1 were lower than those in normal h-CLAT. Therefore, we investigated the effects of the medium and secretion by NHEK cells on THP-1 cells. CD54 and CD86 expression was enhanced in monocultured THP-1 in the medium for keratinized NHEK and in the conditioned medium of keratinized NHEK. The increase in CD54 and CD86 by changes in the medium type was higher than that by the NHEK secretion; therefore, it was found that the medium composition has a large effect on the evaluation index among the experimental parameters in the co-culture system. It is necessary to find the optimal medium for immunotoxicity assessment in the co-culture system.

1. Introduction

Skin sensitization is an allergic reaction that occurs after a single substance or mixture comes into contact with the skin [1], and repeated contact with the skin-sensitizing substance causes local inflammation and irritation on the skin. In recent years, skin sensitization is one of the diseases of high social interest because the accident of skin irritation has occurred not only in the workplace but also in daily life, for example, by products purchased by general consumers. Therefore, it is important to precisely evaluate the skin sensitization of chemical substances. Chemical skin sensitization comprises four key events (KEs) of the adverse outcome pathway (AOP): (i) the covalent binding of electrophilic substances to nucleophilic centers in skin proteins, (ii) inflammatory response by keratinocytes, (iii) dendritic cell (DC) activation, and (iv) T-cell proliferation in the lymph nodes. Alternative methods to predict skin sensitization capabilities have been developed to address these KEs. The KE1 of AOP [2]: Direct Peptide Reactivity Assay (DPRA) and Amino Acid Derivative Reactivity Assay (ADRA), KE2 [3]: KeratinoSensTM and LuSens, KE3 [4]: the human cell line activation test (h-CLAT), U937 cell line activation test (U-SENSTM), and the interleukine-8 reporter gene assay (IL-8 Luc assay). However, it is not possible to completely replace conventional animal testing to predict the skin-sensitizing potential with the single test of these alternative methods because skin sensitization is a complex immune response [5,6]. Therefore, the Organization for Economic Co-operation and Development (OECD) reported the guidance on integrated approaches to testing and assessment (IATA) based on AOP [7]. Many testing strategies combining above alternative methods or other novel methods including in silico methods using the physicochemical index of the chemical substance have been proposed as “defined approaches” (DAs) that form part of IATA of skin sensitization. Some of the reported DAs show at least comparable performance in predicting the Local Lymph Node Assay (LLNA) hazard (sensitizer/non-sensitizer) [8]. Although several studies on the combination of approaches are underway, there are restrictions on the scope of application of chemical substances to alternatives to animal testing, for example, testing of pro-/pre-hapten, lipophilic materials, and those of low solubility are still challenging. Therefore, one of the studies for solving the above problem is an immune-competent in vitro co-culture system as an approach for skin sensitization assessment. It attempts to solve the above problem by observing a series of molecular or cell interactions between KEs within a single method.
To date, co-culture systems combining keratinocytes and THP-1 cells used as a surrogate for DCs in h-CLAT have been reported to be useful as an alternative method for evaluating skin sensitization [9]. For example, Galbiati et al. directly co-cultured the epithelial-like cell line NCTC2544 with THP-1 cells and showed that keratinocytes promote DC maturation [10]. In addition, it was reported that the co-culture system of human epidermal keratinocyte cell line HaCaT and THP-1 cells detected the dependence of the concentration of skin sensitizers and showed a possibility to predict the sensitization intensity [11,12]. In contrast to the above system of homogeneous co-culture of two types of cells, Schellenberger et al. reported that THP-1 cells were placed underneath 3D reconstructed human epidermis (RHE) such as SkinEthic™ (Episkin) and OS-REp (The Phenion® Open Source Reconstructed Epidermis) on cell culture inserts, and the avoidance of co-exposure of both cells will improve testing of less water-soluble chemicals [13]. In the above attempt, ICAM-1 (CD54) and B7-2 (CD86) expression on THP-1 cells were used as the main markers as well as h-CLAT [14,15,16,17].
Activation of immune cells, which are used as markers in skin immunotoxicity assessment, shows a complex response compared to simple cell viability. It has been reported that various factors, such as the pH decrease of the medium [18], lipopolysaccharide (LPS) [19,20], reactive oxygen species (ROS) produced by exposure to the test substance [21] besides a chemical substance, affect the activation of signal pathways involved in the expression of CD54 or CD86. These factors can cause false negative and false positive evaluations in the h-CLAT.
We evaluated skin sensitization using the h-CLAT evaluation criteria in a co-culture system of THP-1 cells and keratinized normal human epidermal keratinocyte (NHEK) cells. Therefore, we collected information on the interaction between NHEK and THP-1 cells to solve biological issues such as medium selection. In the new approaches described above, the test was conducted in the medium for THP-1 cells, whereas in this study, to reduce the stimulation induced by changing the types of medium on both cell types, we used a mixture of two types of media for NHEK and THP-1 cells based on the ratio of the number of seeded cells. In addition, undifferentiated and keratinized NHEK cells were compared to investigate their effects on THP-1 cells. A study using primary mouse epidermal keratinocytes (MEKs) proposed that the expression and inducibility of certain enzyme activities is regulated by the extracellular Ca2+ concentration in the medium and possibly by Ca2+-induced differentiation of MEKs [22]. Therefore, we hypothesized that differences in keratinocyte maturation would fluctuate the magnitude of the effect on THP-1 cells. Collecting data on the effects of media and co-cultured cells on protein expression, which is an indicator of immunotoxicity assessment not only expands the range of application of test substances in h-CLAT, but is also useful for in vitro research that reproduces the in vivo environment by combining multiple cells or tissues such as Organ on a chip [23].

2. Materials and Methods

A schematic diagram of the study is shown in Figure 1. THP-1 cells seeded on a 24-well plate were combined with NHEK cells in an air–liquid interface on a cell culture insert. The effects of not only the chemical substances reached through the epidermal layer but also the secretion from the epidermal layer on THP-1 cell surface protein expression was observed.

2.1. Reagent

2,4-Dinitrochlorobenzene (DNCB, CAS 97-00-7, purity: ≥99%), lactic acid (LA, CAS 50-21-5), and isoeugenol (IE, CAS 97-54-1, purity: ≥98%, mixture of cis and trans) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO, purity: ≥99%) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Saline solution was obtained from Otsuka Pharmaceutical Co. (Tokyo, Japan).

2.2. Cell Culture and Maintenance

The human monocytic leukemia cell line THP-1 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium (RPMI) with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, and 1% (v/v) penicillin-streptomycin solution (all from Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO2. The cells were routinely sub-cultured every two to three days at a density of 1.0–2.0 × 105 cells/mL. Prior to the h-CLAT assay, cells were seeded at 1.0–2.0 × 105 cells/mL and cultured for 48–72 h. The density of the cells was maintained at <5.0 × 105 cells/mL, and the passage number was <30.

2.3. Preparation of Human Epidermis Model

A human epidermis model was prepared using EPI-KIT (JTEC, Aichi, Japan) according to the manufacturer’s protocol. After thawing, 3.5 × 105 NHEK cells in keratinization induction (KI) medium were seeded in the cell culture insert (Falcon® Cell Culture Inserts, pore size 0.4 µm, Corning, NY, USA). Then, the cell culture insert with NHEK cells was placed on a 12-well plate containing KI medium and cultured overnight. After that, the medium on the cell culture insert was removed, and air–liquid interfacial culture was performed for two weeks to stratify the NHEK cell layer. The medium was replaced with fresh medium thrice per week. When NHEK cells were used in an undifferentiated state, NHEK cells were cultured in a medium for proliferating normal human epidermal keratinocytes, HuMedia-KG2 (KG2, Kurabo, Osaka, Japan). After culturing for 48 h for cell adhesion, it was used for testing.

2.4. Preparation of Conditioned Medium from Keratinized NHEK Cells

On days 13 and 14 of air–liquid interfacial culture of NHEK cells, the side surface of the cell culture insert was washed with phosphate buffered saline (PBS, FUJIFILM Wako Pure Chemical Corporation). Then, the cell culture insert with NHEK cells was placed on a 12-well plate containing RPMI medium, KI medium, or the 1:1 mixed medium. After 6, 12, 24, and 36 h, media were collected.

2.5. Treatment of Medium and Chemical in Monoculture and Co-Culture

After washing with PBS, 1.0 × 106 THP-1 cells in 20:7 mixture (vol.) medium of RPMI medium and KI medium were seeded in each well of a 24-well plate. At this time, the amount of medium used was 500 µL for monoculture and 900 µL for co-culture. Increasing concentrations of the test chemicals were prepared at 500 µL for monoculture and 100 µL for co-culture, and both cases had the same final concentration at 1 mL per well. These test chemicals were exposed directly to monocultured THP-1 cells, whereas to co-cultured THP-1 cells via NHEK cells on cell culture inserts, which were attached to each well immediately before exposure.
In the test examining the effect of KI medium and keratinized NHEK cells, 1.0 × 106 THP-1 cells in 1 mL of RPMI medium, KI medium, and 1:1 and 20:7 mixed (vol.) medium of RPMI and KI medium were monocultured or co-cultured with keratinized NHEK cells on a cell culture insert for 24 h. In the test investigating the effects of KG2 medium and co-culturing with undifferentiated NHEK cells, 1.0 × 106 THP-1 cells in RPMI medium, KG2 medium, 1:1 mixed (vol.) medium of RPMI and KG2 medium were monocultured or co-cultured with undifferentiated NHEK cells on a cell culture insert for 24 h.
In the test examining the effects of conditioned medium from keratinized NHEK cells, 1.0 × 106 THP-1 cells were monocultured for 24 h in the conditioned medium described in Section 2.4.

2.6. Analysis of CD54, CD86 Expression Level and Cell Viability

CD54 and CD86 expression and cell viability were analyzed according to the h-CLAT protocol [4]. After culturing for 24 h, the treated cells were collected into sample tubes and centrifuged at 700× g for 3 min. The supernatants were removed by decantation and the cell pellet was blocked with blocking solution [0.01% (w/v) Globulins Cohn fraction II and III, Sigma-Aldrich]. Antibody solutions were prepared using anti-CD86 antibody (BD Pharmingen, San Diego, CA, USA) or anti-CD54 antibody (Dako, Glostrup, Denmark), and FITC-labeled mouse IgG1 (Dako) was added to PBS with 0.1% BSA. The cells were split into three aliquots before staining with the above antibody solutions. After the antibody staining, cells were washed twice with 200 µL PBS supplemented with 0.1% (w/v) BSA and re-suspended in a final volume of 400 µL. Immediately before measuring with a flow cytometer, propidium iodide (PI) solution (Wako Pure Chemical Industries Ltd., Osaka, Japan) was added at the final concentration of 0.625 µg/mL.

2.7. Data Analysis and Criteria

The expression levels of cell surface antigens were measured using flow cytometry with a FACS Calibur (Becton Dickinson, San Jose, CA, USA). The FITC acquisition channel (FL-1) and the PI acquisition channel (FL-3) were adjusted for optimal detection of the FITC fluorescence signal and PI fluorescence signal, respectively. Dead cells were stained and visualized with PI. The total number of living cells used for evaluation is 10 × 104. The viability and the mean fluorescence intensity (MFI) of viable cells of each sample were determined. The relative fluorescence intensity (RFI) is expressed by the following formula as an index of the expression levels of CD54 and CD86 for each concentration of all test substances.
R F I ( % ) = ( MFI   of   chemical − treated   cells ) − ( MFI   of   test   chemical − treated   isotype   control   cells ) ( MFI   of   solvent − treated   cells ) − ( MFI   of   solvent − treated   isotype   control   cells ) × 100
MFI = Geometric mean fluorescence intensity.
For each concentration of the test material, cell viability was recorded from the isotype control cells. The h-CLAT criteria (CD54 ≥ 200% and/or CD86 ≥ 150%) were used to evaluate the results according to the protocol of h-CLAT [4].

2.8. Statistical Analysis

In this study, cell-based assays were performed in triplicate and the data presented are expressed as the mean ± standard deviation (SD). The statistical significance of differences in the RFIs of CD54 and CD86 between monocultured THP-1 cells and co-cultured THP-1 cells and between THP-1 cells monocultured in RPMI medium and THP-1 monocultured in KI, KG2, and mixed media was evaluated using one-way ANOVA followed by Dunnett’s test. A p value < 0.05 was considered significant. All statistical analyses of recorded data were performed using EZR [24], which is a graphical user interface for R (The R Foundation for Statistical Computing, version 3.3.0).

3. Results

3.1. Comparison of Normal h-CLAT and Co-Culture System h-CLAT

The expression of CD54 and CD86 in THP-1 cells monocultured and co-cultured with keratinized NHEK cells as a skin model was evaluated. First, according to the h-CLAT protocol of the concentration setting test, the cell viability 75% (CV75) doses for each chemical were determined. CV75 doses for DNCB, LA, and IE of monocultured THP-1 cells were 5.1 mg/mL, 3.1 × 103 mg/mL, and 1.1 × 102 mg/mL, respectively. In contrast, CV75 doses for DNCB and LA under co-culture conditions were 6.1 mg/mL and 3.1 × 103 mg/mL, respectively. The CV75 dose for IE in the co-culture condition could not be determined because THP-1 cell viability was maintained over 80% under the examined conditions (data not shown).
Based on CV75 doses, the expression of CD54 and CD86 in THP-1 cells after exposure to each chemical at eight concentrations (common ratio of 1.2) was measured. In the case of IE in the co-culture condition, THP-1 cells were exposed to IE solution of CV75 dose for IE under monoculture conditions (111 µg/mL) and seven higher concentrations (common ratio of 1.2). DNCB treatment upregulated CD54 and CD86 beyond the h-CLAT criteria (CD54 ≥ 200% and/or CD86 ≥ 150%) (Figure 2A). THP-1 cells co-cultured with keratinized NHEK cells also showed a positive response using h-CLAT criteria (Figure 2B). However, the RFIs of CD54 and CD86 in co-cultured THP-1 cells were lower than that of the normal h-CLAT (Figure 2A). For LA, the RFIs of CD54 and CD86 were under 200 and under 150, respectively, on both mono-cultured (Figure 3A) and co-cultured (Figure 3B) THP-1 cells. IE showed a negative response in the normal h-CLAT (monocultured THP-1 cells, Figure 4A), whereas IE upregulated CD54 and CD86 beyond the h-CLAT criteria in the THP-1 cells co-cultured with keratinized NHEK cells (Figure 4B). It was shown that co-culture could evaluate IE, which is a prohapten.

3.2. Effects of Medium and Co-Culturing with NHEK Cells on CD54 and CD86 Expression Levels of THP-1 Cells

The effect of medium and co-culture with keratinized NHEK cells on the expression of CD54 and CD86 in THP-1 cells was analyzed. First, the expression of CD54 and CD86 in THP-1 cells monocultured in RPMI medium, KI medium, and 1:1 and 20:7 mixed (vol.) medium of RPMI medium and KI medium was examined. Figure 5 shows the effects of each medium on the expression of CD54 and CD86 in monocultured THP-1 cells. In KI medium, the expression levels of CD54 and CD86 were increased two-fold and three-fold, respectively, compared to the RPMI medium, which was a normal condition of h-CLAT (Figure 5A,B, monoculture). The reduction of the KI medium ratio to 50% (1:1 mixed medium) and 35% (20:7 mixed medium) did not suppress the expression of CD54 and CD86. In the case of the co-culture system with keratinized NHEK cells, the expression levels of CD54 and CD86 in THP-1 cells in RPMI medium were increased by 2-fold and 2.5-fold, respectively, compared to the normal h-CLAT conditions (monocultured THP-1 cells in RPMI). In the case of KI medium and 1:1 and 20:7 mixed (vol.) medium of RPMI and KI medium, co-culture with keratinized NHEK cells did not significantly increase the levels of CD54 and CD86. However, there was a trend for increasing the expression of CD54 (Figure 5A,B, co-culture). It was shown that the protein expression levels on THP-1 cells fluctuate under the influence of co-cultured NHEK and the medium.

3.3. Effects of KG2 Medium and Co-Culturing with Undifferentiated NHEK Cells

The effect of medium and co-culture with undifferentiated NHEK cells on the expression of CD54 and CD86 in THP-1 cells was analyzed. Figure 5 shows the effects of each medium on the expression of CD54 and CD86 in monocultured THP-1 cells. In KG2 medium, the expression level of CD54 was increased three-fold compared to that in the RPMI medium (Figure 5C,D, monoculture). In the case of 1:1 mixed (vol.) medium of RPMI and KG2 medium, although there was a trend for increasing the expression of CD54 (Figure 5C, co-culture), the difference was not statistically significant.
Co-culture with undifferentiated NHEK cells increased the expression of CD54 in RPMI, KG2, and 1:1 mixed medium although the difference was not statistically significant. We have shown that the effect to THP-1 cells varies depending on whether the co-cultured NHEK is undifferentiated or keratinized.

3.4. Effects of Conditioned Medium from Keratinized NHEK Cells on the Expression of CD54 and CD86

RPMI, KI, and 1:1 mixed medium in which NHEK cells were cultured for 6, 12, 24, and 36 h were collected. THP-1 cells were cultured in conditioned media for 24 h, and the expression levels of CD54 and CD86 were measured. The expression levels of CD54 and CD86 were increased in RPMI (Figure 6). In RPMI medium, the expression of CD54 and CD86 decreased as the preparation time of the conditioned medium increased. In KI and 20:7 mixed medium, the expression of CD54 and CD86 did not change as the preparation time of the conditioned medium increased (Figure 6). When co-culturing with NHEK, it was found that the increase in the protein expression levels on THP-1 cells was suppressed by acclimatizing NHEK in RPMI medium.

4. Discussion

Since the epidermis model used in this study uses an in-house model instead of the existing commercial model, this quality evaluation was performed prior to the sensitization test. It was confirmed that this model meets the criteria for barrier function described in the standardized test method (TG439) for skin irritation tests using the three-dimensional cultured epidermis model shown by the OECD [25]. In addition, the quality of the epidermis model used was ensured by measuring the transdermal electrical resistance (TEER), which is an index of barrier function, and observing the cell layer by microscopic observation after HE staining of the immobilized section. The materials and methods of the experiment and the details of the results are described in Supplemental Materials.
Observing crosstalk between skin cells and immune cells is necessary for predicting skin sensitization of chemical compounds. However, it was considered that cell functionality such as the immune response used for an evaluation index is very sensitive to culture conditions. The main purpose of this study was to obtain expertise about the effects of the co-culture system on the evaluation index of h-CLAT.
First, we confirmed that DNCB was evaluated as positive (Figure 2B) and LA as negative (Figure 3B) in co-culture h-CLAT. This study demonstrated that IE, which was evaluated as negative in normal h-CLAT (Figure 4A), was evaluated as positive in the co-culture system (Figure 4B). The difference between monoculture and co-culture in IE results can be explained by the metabolization of IE by NHEK cells. For a pre- or pro-hapten, the formation of reactive hapten by spontaneous air-oxidation (prehapten) or enzyme-mediated activation (prohapten) is needed to obtain the skin sensitization potential [26,27]. It has been reported that keratinocytes express metabolic enzymes such as the cytochrome P450 (CYP) superfamily [28]. Similar to the report by Esks et al. that the HaCaT/THP-1 cell co-culture system improved the evaluation of pro-/pre-haptens [12], it was considered that the effect of metabolism by NHEK cells was observed in this study as well. It is considered that co-culture system allows to observe the effects of the parent compound and the metabolite simultaneously. In contrast, it is difficult to examine these separately in a co-culture system; therefore, it is important to break down the pathways individually and examine them as in the study in which THP-1 cells were treated with prohapten bioactivated by S9 fraction [29]. Concluding from the results of Figure 1 to Figure 4, we showed the possibility of evaluating the effect of prohapten, which acquires sensitization for the first time by being metabolized by the epidermis model. Therefore, we also emphasize the importance of developing a co-culture system.
While demonstrating its usefulness for predicting skin sensitization, co-cultured THP-1 cells showed a lower rate of increase in CD54 and CD86 expression levels upon DNCB exposure (Figure 2B) than monocultured THP-1 cells (Figure 2A). In the control cells in the co-culture system (THP-1 cells co-cultured with NHEK cells in 20:7 mixed medium), the MFI values of CD54 and CD86 were higher than those in the control cells in normal h-CLAT (THP-1 cells monocultured in RPMI medium). Therefore, it was speculated that the difference between the control cells and the DNCB-treated cells was difficult to observe in the co-culture system. The reason was that the expression levels of CD54 and CD86 were already increased in the control cells owing to the 20:7 mixed medium and the secretion from NHEK cells, so that the expression levels in chemical (DNCB)-treated cells appear relatively lower. Therefore, the effects of media and NHEK cells on the expression levels of CD54 and CD86 on THP-1 cells were examined in detail.
KI medium, which is a medium for induction keratinization of NHEK cells, enhances the expression of CD54 and CD86 in monocultured THP-1 cells. There was no significant difference in the level of upregulation of CD54 and CD86 between 100% KI medium and 50% (1:1 mixed medium) or 35% (20:7 mixed medium), that is, decreasing the mixing ratio of KI medium up to 35% did not suppress the up-regulation. The detailed components of the KI medium are not disclosed. KI medium has a higher concentration of calcium ions, which is a requirement for induction of keratinization, than general media. Rossol et al. provided evidence that increased extracellular Ca2+ ions can activate the NLRP3 inflammasome, which plays a role in controlling skin sensitization [30] in monocytes [31]. Mitachi et al. reported that the NLRP3 inflammasome is involved in the upregulation of CD54 expression in THP-1 cells [32]. Corresponding to these published data, our results revealed that high Ca2+ in the KI medium may enhance CD54 expression in THP-1 cells. In the future, it will be necessary to verify the identification of the factors of the conditioned medium. The concentration of Ca2+ in the medium, which greatly affects the keratinocyte keratinization, is a factor that influences the state of keratinocyte, and it can be expected that it will lead to changes in the environment surrounding THP-1 cells.
KG2 medium, which is a medium that maintains the undifferentiated state of NHEK cells, enhances CD54 expression in THP-1 cells, whereas it suppresses CD86 expression. KG2 medium is a serum-free medium; therefore, the serum concentration in the medium was considered to be involved in the change of these expression levels. ROS cannot be alleviated in serum-free medium, and its involvement in differentiation, metabolic function, viability, and proliferation of THP-1 cells has been reported [33]. To evaluate skin sensitization in a system using the activation of THP-1 cells as an evaluation index, attention should be paid to serum volume. The increase in CD54 expression and the decrease in CD86 expression were suppressed by 50% of KG2 medium (1:1 mixed medium) rather than 100%; therefore, lowering the ratio of KG2 medium or adjusting the serum volume was considered to be an effective way to reduce the impact of the evaluation index.
It is known that keratinocytes produce several cytokines mainly involved in immunity and inflammation, such as TNF-α [34], IL-10 [35], GM-CSF, and IL-1 [36]. These cytokines cause epidermal Langerhans cells to increase a6 integrin [37], migrate to lymph nodes [38], induce immune tolerance [39], and enhance and assist the ability of antigen-presentation [40,41], respectively. Primary cells are expected to maintain original functions closer to living tissue than cell lines, for example, the ability to produce a variety of cytokines and metabolic enzymes [42]; therefore, NHEK cells were used in this study. The expression levels of CD54 and CD86 were compared between THP-1 monoculture and co-culture using RPMI medium and increased by co-culturing with keratinized NHEK cells. It was suggested that RPMI-stimulated keratinized NHEK cells release secretions that enhance the expression of CD54 and CD86. The results showed that the effect of KI medium on THP-1 cells was greater than that of keratinized NHEK cell secretions.
The expression levels of CD54 and CD86 were compared between THP-1 cell monoculture and co-culture using RPMI medium and increased by co-culturing with undifferentiated NHEK cells. According to the comparison of the results, the co-cultured NHEK cells showed that the upregulation of CD54 and CD86 was more suppressed in the keratinized state than in the undifferentiated state. Keratinization was considered to reduce the amount of secretions that affects THP-1 cells. It was speculated that this was because it became stable as it “matured” [43]. As a further hypothesis, one of the differences between the undifferentiated state and the keratinized state of NHEK cells is the number of epidermal basal cells. Epidermal basal cells proliferate in KG2 medium, whereas proliferating cells and keratinized cells coexist in KI medium and the number of epidermal basal cells is expected to be lower after two weeks of air-liquid interface culture. Therefore, it was considered that the epidermal basal cells produce secretions that enhance the expression of CD54 and CD86. In addition, when co-cultured with undifferentiated NHEK cells, the expression levels of CD54 and CD86 in THP-1 cells varied widely, and there seemed to be considerable uncertainty about predicting sensitization.
It was considered that reducing the stimulation of the keratinized NHEK cells with RPMI medium suppressed the production of factors that affect the expression of CD54 and CD86 in THP-1 cells. Therefore, we investigated the acclimatization of NHEK cells to RPMI medium. For the collected RPMI medium, in which only NHEK cells were cultured for 6, 12, 24, and 36 h, the longer the culture time of NHEK cells, the lower the expression levels of CD54 and CD86 in THP-1 cells. These results suggest that the amounts of factors produced by NHEK cells decrease over time. Keratinocyte growth factor (KGF) produced by fibroblasts is involved in keratinocyte proliferation; therefore, the primary keratinocytes can be maintained by culturing with feeder layer or a conditioned medium with fibroblasts [44]. Medium changes can be stressful for keratinocyte cultures, given that an environment containing the necessary nutrients or factors is required. Undernourishment stress, such as deficiency of pantothenic acid, a water-soluble vitamin, arrests cell proliferation and promotes differentiation [45], and keratinocytes, either constitutively or upon stimulation, express IL-1, -6, -7, -8, -10, -12, -15, -18, and -20 [46] and TGF-β1 [47]. As an interpretation of our results, it was considered that the stress on NHEK cells caused by the change to RPMI medium was alleviated during 36 h of culture, and the secretion of factors that enhance the expression of CD54 and CD86 in THP-1 cells was decreased. It is generally known that signal proteins generated by cells are decomposed over time to maintain a dynamic equilibrium state [48].
We have revealed the effect of the medium. However, we have not yet optimized it to reproduce the in vivo sensitization response. It is necessary to verify whether it is optimal to increase or decrease the expression levels of CD54 and CD86 by changing the medium. We have shown that the base (CD54 and CD86 expression levels under control conditions) increases with changes in the medium, which leads to a decrease in evaluation sensitivity. First, we are thinking that it is necessary to develop the co-culture evaluation system to suppress the rise of the base by mixing the medium. In conclusion, this study showed that when producing a co-culture system for skin sensitization evaluation, it is necessary to select a medium that is optimal for culturing both cells and does not affect the functionality of the cells used as an evaluation index. In addition, it was reported that the medium stimulates THP-1 and NHEK cells, and through cell–cell interaction, the evaluation index of skin sensitization fluctuated in THP-1 cells. It is important to select a medium for THP-1 cells as well as NHEK cells that assist THP-1 cells in the co-culture system. The knowledge gained from this study is used to support the development and evaluation of novel alternative approaches for the evaluation of skin-sensitizing chemicals.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app12126207/s1, Figure S1: Transition of TEER value of epidermis model for each culture day; Figure S2: HE staining of stratified 3D cultured epidermis model on insert membrane after 14 days of air-liquid interfacial culture. References [25,49] are cited in the supplementary materials.

Author Contributions

Conceptualization, H.I. and J.F.; methodology, H.I.; investigation, Y.S. and H.T.; writing—original draft preparation, Y.S.; writing—review and editing, K.I.; supervision, H.I.; project administration, H.I.; funding acquisition, H.I and J.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partly supported by a grant from the Long-range Research Initiative (LRI, No. 17_PT01-01) by the Japan Chemical Industry Association (JCIA).

Institutional Review Board Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic view of the workflow of the h-CLAT procedure in co-culture system.
Figure 1. Schematic view of the workflow of the h-CLAT procedure in co-culture system.
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Figure 2. CD54 and CD86 expression induced by DNCB on THP-1 cells in monocultured (A) and co-cultured with keratinized NHEK cells (B). DNCB solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (A), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (B). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
Figure 2. CD54 and CD86 expression induced by DNCB on THP-1 cells in monocultured (A) and co-cultured with keratinized NHEK cells (B). DNCB solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (A), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (B). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
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Figure 3. CD54 and CD86 expression induced by LA on THP-1 cells in monocultured (A) and co-cultured with keratinized NHEK cells (B). LA solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (A), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (B). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
Figure 3. CD54 and CD86 expression induced by LA on THP-1 cells in monocultured (A) and co-cultured with keratinized NHEK cells (B). LA solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (A), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (B). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
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Figure 4. CD54 and CD86 expression induced by IE on THP-1 cells in monocultured (A) and co-cultured with keratinized NHEK cells (B). IE solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (A), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (B). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
Figure 4. CD54 and CD86 expression induced by IE on THP-1 cells in monocultured (A) and co-cultured with keratinized NHEK cells (B). IE solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (A), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (B). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
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Figure 5. The expression levels of CD54 and CD86 on THP-1 cells were increased by changing the medium or co-culturing with NHEK compared to the single culture. The effect of medium on cell viability and basal CD54 and CD86 expression of mono-cultured and co-cultured THP-1 cells. Cell viability and expression levels of CD54 (A,C) and CD86 (B,D) on THP-1 mono-cultured or co-cultured with keratinized NHEK cells (A,B) and undifferentiated NHEK cells (C,D) in various kinds of medium were analyzed using flow cytometry (mean ± SD, n = 3). RFI, relative fluorescence intensity; KG2, normal human epidermal keratinocyte growth medium supplemented with a set of growth additives, KI, keratinization induction medium; 20:7 mix, mixing ratio was RPMI:KI = 20:7. *, **, ***: Statistically significant from control (monoculture RPMI) or between indicated sample pair by Dunnett’s post-hoc test. * p < 0.05. ** p < 0.01. *** p < 0.005. N.S.: not significant. The black (left) and gray (right) dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
Figure 5. The expression levels of CD54 and CD86 on THP-1 cells were increased by changing the medium or co-culturing with NHEK compared to the single culture. The effect of medium on cell viability and basal CD54 and CD86 expression of mono-cultured and co-cultured THP-1 cells. Cell viability and expression levels of CD54 (A,C) and CD86 (B,D) on THP-1 mono-cultured or co-cultured with keratinized NHEK cells (A,B) and undifferentiated NHEK cells (C,D) in various kinds of medium were analyzed using flow cytometry (mean ± SD, n = 3). RFI, relative fluorescence intensity; KG2, normal human epidermal keratinocyte growth medium supplemented with a set of growth additives, KI, keratinization induction medium; 20:7 mix, mixing ratio was RPMI:KI = 20:7. *, **, ***: Statistically significant from control (monoculture RPMI) or between indicated sample pair by Dunnett’s post-hoc test. * p < 0.05. ** p < 0.01. *** p < 0.005. N.S.: not significant. The black (left) and gray (right) dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 ≥ 200%, CD86 ≥ 150%).
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Figure 6. The effect of conditioned medium on cell viability and basal CD54 and CD86 expression on monocultured THP-1 cells. THP-1 cells were cultured in the conditioned medium in which keratinized NHEK cells were cultured for 6, 12, 24, and 36 h, and cell viability and their expression of CD54 and CD86 were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. * p < 0.05, significantly different from the control (“normal”) (by ANOVA followed by Dunnett’s test).
Figure 6. The effect of conditioned medium on cell viability and basal CD54 and CD86 expression on monocultured THP-1 cells. THP-1 cells were cultured in the conditioned medium in which keratinized NHEK cells were cultured for 6, 12, 24, and 36 h, and cell viability and their expression of CD54 and CD86 were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI ± SD for three individual experiments are shown. * p < 0.05, significantly different from the control (“normal”) (by ANOVA followed by Dunnett’s test).
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Sawada, Y.; Tsukumo, H.; Fukuda, J.; Iijima, K.; Itagaki, H. Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT). Appl. Sci. 2022, 12, 6207. https://doi.org/10.3390/app12126207

AMA Style

Sawada Y, Tsukumo H, Fukuda J, Iijima K, Itagaki H. Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT). Applied Sciences. 2022; 12(12):6207. https://doi.org/10.3390/app12126207

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Sawada, Yuka, Hanae Tsukumo, Junji Fukuda, Kazutoshi Iijima, and Hiroshi Itagaki. 2022. "Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT)" Applied Sciences 12, no. 12: 6207. https://doi.org/10.3390/app12126207

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