Development of a multi-analysis model using an epithelial-fibroblast co-culture system as an alternative to animal testing

Article information

Environ Anal Health Toxicol. 2024;39.e2024024

Publication date (electronic) : 2024 September 27

doi : https://doi.org/10.5620/eaht.2024024

Min-Ju Kim ¹

, Hee-Sung Hwang ¹

, Jee Hoon Choi ¹

, Eun-Seon Yoo ¹

, Mi-Im Jang ¹

, Juhee Lee ³

, Seung Min Oh ¹^,²^,

¹Department of Bio-application toxicity, Hoseo University, Asan, Republic of Korea

²Department of Animal Health and Welfare, Hoseo University, Asan, Republic of Korea

³Department of ICT Automotive Engineering, Hoseo University, Asan, Republic of Korea

^*Correspondence: ohsm0403@hoseo.edu

Received 2024 July 8; Accepted 2024 September 13.

Abstract

The evaluation of respiratory chemical substances has been mostly performed in animal tests (OECD TG 403, TG 412, TG 413, etc.). However, there have been ongoing discussions about the limited use of these inhalation toxicity tests due to differences in the anatomical structure of the respiratory tract, difficulty in exposure, laborious processes, and ethical reasons. Alternative animal testing methods that mimic in vivo testing are required. Therefore, in this study, we established a co-culture system composed of differentiated epithelial cells under an air-liquid interface (ALI) system in the apical part and fibroblasts in the basal part. This system was designed to mimic the wound-healing mechanism in the respiratory system. In addition, we developed a multi-analysis system that simultaneously performs toxicological and functional evaluations. Several individual assays were used sequentially in a multi-analysis model for pulmonary toxicity. Briefly, cytokine analysis, histology, and cilia motility were measured in the apical part, and cell migration and gel contraction assay were performed by exposing MRC-5 cells to the basal culture. First, human airway epithelial cells from bronchial (hAECB) were cultured under air-liquid interface (ALI) system conditions and validated pseudostratified epithelium by detecting differentiation-related epithelial markers using Transepithelial Electrical Resistance (TEER) measurement, Hematoxylin and Eosin (H&E) staining, and immunocytochemistry (ICC) staining. Afterward, the co-culture cells exposed to Transforming growth factor-beta 1 (TGF-β1), a key mediator of pulmonary fibrosis, induced significant toxicological responses such as cytotoxicity, cell migration, and gel contraction, which are wound-healing markers. In addition, cilia motility in epithelial cells was significantly decreased compared to control. Therefore, the multi-analysis model with a 3D epithelial-fibroblast co-culture system is expected to be useful in predicting pulmonary toxicity as a simple and efficient high-throughput screening method and as an alternative to animal testing.

Keywords: 3D epithelial-fibroblast co-culture system; Multi-analysis; Primary Human Airway Epithelial Cells from Bronchi (hAECB); Lung Fibroblast Cells (MRC-5); Toxicological and Functional Evaluation

Introduction

Inhalation is the main route of exposure for humans, and inhalation exposure to various chemicals can cause respiratory diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), asthma, and lung cancer. The evaluation of chemical substances on respiratory diseases has been mostly performed in animal experiments [1]. The OECD Test Guidelines (TG) for inhalation toxicity testing include standards for acute (TG 403, 433, 436), 28-day (TG 412), and 90-day (TG 413) testing [2]. However, there are growing concerns that animal studies do not always optimally predict human responses to inhaled substances, and there is growing public concern about animals used for research purposes. Therefore, efforts are being actively made to find alternatives that can reduce, refine, and replace these animal model experiments (the 3Rs) [3]. Recently, as an alternative, in vitro three-dimensional (3D) culture models using primary lung cells for inhalation toxicity experiments are being actively studied [4].

In the lung, the respiratory epithelium is the first tissue with which inhaled substances directly interact and is the major focus in cell culture models for inhalation toxicity studies. This airway epithelium is lined by a pseudostratified epithelium composed of a variety of cell types, including mucus-producing goblet cells, ciliated cells, non-ciliated club cells, and basal cells, which acts as a physical and immunological barrier protecting the subepithelial tissue [5, 6]. This can be accomplished in vitro through air-liquid interface (ALI) conditions, which are often applied to airway epithelial cell culture [7]. To be appropriate for in vitro inhalation toxicology studies, an in vitro epithelial cell culture system must mimic the function of the lung epithelium in the best possible way. Therefore, various co-culture models are being developed, including epithelial cells, fibroblasts, endothelial cells, or airway smooth muscle cells, which are the main constituent cells of lung tissue, and macrophages or dendritic cells, which are immune cells [3].

The lung epithelium acts as both a barrier and source of pathogenic signaling and initiates the process of fibrosis [8, 9, 10]. The principal initiating process in fibrosis involves crosstalk between damaged epithelium and fibroblasts [11, 12]. Damaged epithelium activates fibroblasts by secreting various cytokines, including interleukin (IL)1- α, IL-4, IL-6, and tumor necrosis factor-alpha (TNF-α). It is known to induce differentiation into myofibroblasts, promoting excessive deposition of extracellular matrix (ECM) and abnormal accumulation of collagen [13, 14, 15].

In this study, we established an in vitro co-culture model which composed of a differentiated epithelial cell and fibroblast and applied multiplex analysis as a high-throughput screening method for the evaluation of pulmonary toxicity. In the 3D epithelial-fibroblast co-culture system exposed to TGF-β1, a key mediator of pulmonary fibrosis was simultaneously evaluated both toxicological and functional toxic responses for pulmonary. Toxicological evaluation included assessment of cytotoxicity, oxidative stress, and inflammation-related cytokine expression, and functional evaluations included assessment of cilia morphology, cilia motility, cell migration, and collagen contraction. Therefore, we suggested that a multi-analysis model using 3D epithelial-fibroblast co-culture system can be a useful tool as an alternative method of animal experiments for evaluating pulmonary toxicity.

Materials and Methods

A three-dimensional (3D) Air-Liquid Interface (ALI) epithelial-fibroblast co-culture system

To assess the toxic response in the lung, we used an 3D epithelial-fibroblast co-culture system. Briefly, hAECB cells were differentiated under ALI conditions in transwell filters in 24-well plates for over 4 weeks. At this time, the differentiated cells were transferred to a 24 well plate with MRC-5 cells (8 × 10⁴ cells/well) seeded on the bottom.

The primary human airway epithelial cells from bronchi (hAECB), isolated from human lung biopsies, were purchased from Epithelix Sarl (Swizerland). Cryopreserved cells (Batch Number 02AB0940, Epithelix, Swizerland) were cultured and differentiated according to the manufacturer's instructions. Briefly, cells were expanded in T75 flask (cat. 70075, SPL, Pocheon, Korea) using an “expansion medium” consisting of Pneumacult^TM-Ex Plus medium (cat.05040, Stemcell Technologies). The hAECB cells were cultured at 37 °C and 5 % CO₂, changing the medium every other day. Prior to cell seeding, filters of 24-transwell plates (Cat. 3470, Corning, USA) were precoated with 100 μL each of 0.33 mg/mL rat tail collagen I (Cat. 354236, Corning, USA) for 45 min at 37 °C and 5 % CO₂. Air-liquid interface (ALI) conditions were initiated 3 - 5 days after seeding (3.3 x 10⁴ cells/well) by completely aspirating “expansion medium” from the apical compartment and replacing it with the “ALI medium; PneumaCult^TM-ALI basic medium (Cat. 05001, Stemcell Technologies, USA)” containing supplement agents (heparin, hydrocortisone, and maintain supplements). After hAECB cells were differentiated for 4 weeks under ALI conditions, mucus production, and ciliary beating could be observed.

Human fibroblasts (MRC-5) were purchased from the Korea Cell Line Bank (KCLB No. 10171, Korea Cell Line Bank, Korea) and cultured in MEM supplemented with 10 % fetal bovine serum (Lot. 29821001, Corning, USA), 1 ％ penicillin (100 units/ml)/streptomycin (100 μg/ml) at 37 °C, 5 % CO₂ in a Thermo Scientific incubator.

Barrier Integrity of human airway epithelial cells from bronchi (hAECB) cells cultured at the Air-Liquid Interface

The integrity of the airway barrier in a bronchial ALI co-culture system was confirmed by Transepithelial Electrical Resistance (TEER), Hematoxylin and Eosin (H&E) staining, and immunocytochemistry (ICC) staining. TEER was measured weekly using an EVOM 2 epithelial voltmeter (EVOM 2, World Precision Instruments, USA) after removing excess mucous by 1 × DPBS (Cat.LB001-02, WELGENE, USA). TEER (Ohm × cm²) was calculated by multiplying the NET resistance by the surface area of the transwell membrane (0.33 cm²). Imaging for H&E and ICC analysis was performed using an inverted microscope (CX23RTFS2, Olympus, Japan) and laser confocal microscope (LSM880, Carl Zeiss Microscopy, Germany), respectively.

Cell viability test by WST-1 assay and Intracellular reactive oxygen species by DCF-DA

To study cytotoxicity and ROS formation, epithelial-fibroblast co-culture system were treated with TGF-β1 (cat. T7039, Sigma-Aldrich, US) at a concentration 100 ng/ml. The hAECB cells on the apical side and the MRC-5 cells on the basolateral side of the insert were incubated with CellVia solution for WST-1 assay (LF-EZ1001A, AbFRONTIER, USA) for 30 to 90 min and then absorbance (450 nm) was measured using a cell microplate reader (TECAN, Zurich, Switzerland).

To measure the increase of ROS, each well was loaded with DCF-DA (Cat. D399, Thermo Scientific, USA) for 30 minutes and the fluorescence signal was measured using a fluorescence microscope (U-HGLGPS, Olympus, Japan). ROS formation was calculated in the area of the green fluorescent area by image J (v.1.54g).

Cytokine measurement by ELISA in hAECB cell

The cytokines (TNF-α, IL-1α, IL-4 and IL-6, LTGM100, R&D Systems) for fibrotic inflammatory responses were analyzed with apical solution exposed to TGF-β1 in 3D epithelial-fibroblast co-culture system via Luminex® 100/200^TM according to the manufacturer's protocol of Luminex Multiplex assay (RND-LXSAHM-07, R&D Systems, USA). The measured fluorescence wavelength of the laser in the equipment is 525 nm.

Cilia motility of differentiated ciliated epithelial cells

Cilia and mucociliary clearance are the most important physiological defense mechanisms of the airway epithelium [16]. To compare changes in ciliary in response to TGF-β1, we analyzed the ciliary beating area of hAECB cells differentiated under ALI conditions for more than 4 weeks. For the evaluation of ciliary beating areas, images were taken using a 10X objective on an Olympus CKX53 microscope and a fluorescent color IMT Scan camera (IMT Scan, iSolution, Germany) (FOV ≈ 877 μm by 660 μm). To maintain temperature during imaging, hAECB cells were imaged with the bottom of the transwell plate filled with medium preheated to 37 °C in an incubator. The acquired images were evaluated by extracting nine locations in a cross shape at 150 x 150-pixel size at 30 fps, and the application for image capture and analysis was performed using MATLAB R2024a (MATLAB R2024a, MathWorks Inc, USA). The analysis algorithm for cilia beat area measurement used an open-source algorithm developed in assessment of motile ciliary coverage and beat frequency in in vitro cell culture tissues [17]. This algorithm used a Fast Fourier Transform (FFT) of the periodicity variation (autocorrelation) over time for each pixel in the extracted image to obtain the power spectral density (PSD) used for ciliary beat area measurement, which is then compared and analyzed.

Functionality of fibroblast through cell migration and collagen gel contraction

MRC-5 cells (6 × 10⁴ cells) seeded on the basolateral side using culture inserts (Cat. 80209, ibidi GmbH, Germany) were used for cell migration. When the cells reached confluence, culture-inserts were removed. The hAECB cells exposed to TGF-β1 were then transferred to well plates previously prepared with MRC-5 cells. After 9 h, the migration of MRC-5 cells was observed through a microscopic examination (CKX 53, Olympus, Japan) and assessed by quantifying the area of the insert cap by Image J (v.1.54g).

A three-dimensional collagen gel contraction model [18] was used to assess the contractility of MRC-5 cells. MRC5 cells in serum-free 4X MEM medium (3x10⁵ cells/ml) were mixed with 3 mg/ml collagen type 1 rat tail (Corning) and distilled water and incubated at 37 °C for 2 h to harden the gel. And then, 300 μL of the conditioned medium in the basal area of hAECB cells treated with TGF-β1 (100 ng/ml) was added to the well plate containing fibroblast gel. The gel contractility was checked with a microscope (SZ61, Olympus, Japan), and measured the gel area by image J (v.1.54g). Gel contraction was quantified by calculating the difference between the initial gel area and the gel area after 48 h as a percentage.

Statistical methods

Each assay was performed at least three times. SigmaPlot 12.0 (Systat Software Inc., IL, USA) and Excel 2019 (Microsoft, WA, USA) were used for data analysis. Data were expressed as mean ± standard deviation. Statistical differences between each group were evaluated by two-sided Student's test. Statistical significance was accepted at **p < 0.01 and *p < 0.05.

Results

Confirmation of differentiation of Human Airway Epithelial Cells (hAECB) under ALI conditions

The epithelium barrier integrity and characterization were evaluated to confirm complete differentiation in hAECB cells under ALI conditions. The ALI method is advantageous to promote cell differentiation and optimize the morphological and histological characteristics of airway epithelium cells [19]. The in vivo bronchiolar epithelium is characterized by intercellular tight junctions granting a protective physical barrier between the bronchial lumen and the underlying tissue [20]. In this study, the TEER value of hAECB differentiated under ALI conditions was stably maintained above 400 Ω.cm² for 4 weeks (Fig. 1a). The morphology of fully differentiated pseudostratified epithelial layer and ciliated epithelial cells was confirmed by H&E staining (Fig. 1b). Additionally, MUC5AC, a marker for goblet cells, was clearly detected in confocal immunofluorescence microscopy (Fig. 1c).

Figure 1.

Differentiation of Normal Human Bronchial Epithelial Cells under ALI : After cultured hAECB cells in PneumaCultTM-ALI media for 4 weeks at ALI conditions, differentiated pseudostratified epithelial layer was confirmed. (a) The TEER value measured under ALI conditions maintained above 400 Ω.cm² for 4 weeks. (b) Hematoxylin & eosin (H&E) staining of hAECB cells differentiated for over 4 weeks. (c) Immunofluorescence staining of the nuclear (DAPI, blue), goblet cells (MUC5AC, green) of hAECB cells differentiated for over 4 weeks.

Multi-analysis model

We designed an in vitro co-culture system utilizing multiplex analysis as a high-throughput screening method to enhance the predictability and efficiency of toxicity assessment (Fig. 2). Through this model, we simultaneously conducted toxicological and functional evaluations.

Figure 2.

Schematic of multi-analysis model. Schematic representation of the epithelial-fibroblast co-culture system and multiplex analysis. Epithelial cells are seeded (day 0), grown (days 3–5), and differentiated (days 7– 28). After epithelial cells were completely differentiated, 3D Epithelial-fibroblast co-culture system was finally established by co-culturing with fibroblasts. TGF-β1 was then treated with it for 3 h and multi-analysis was performed. Cytokine analysis, cell viability, TEER measurement, histological confirmation, and ciliary motility measurement were performed on apical epithelial cells, and cell viability, ROS generation, cell migration, and collagen gel contraction were performed on basal side fibroblasts.

Toxicological evaluation using epithelial-fibroblast co-culture system

To investigate physiological epithelial disruption of the hAECB cells by specific stimuli, cells were exposed to the indicated concentration of TGF-β1, a key disease mediator in idiopathic pulmonary fibrosis (IPF) [21]. In the co-culture system, hAECB cells in the apical region were exposed to TGF-β1 (100 ng/mL) for 3 h, and MRC-5 cells were seeded in the basal side to be affected by TGF-β1 indirectly. As shown in Fig. 3 (Fig 3a; hAECB cells, Fig 3b; MRC-5 cells), both hAECB cells (in apical) and MRC-5 cells (in basal) exposed to TGF-β1 have a significant decrease in cell viability by approximately 10 %.

Figure 3.

Toxicological evaluation using 3D Epithelial-fibroblast co-culture system: The 3D Epithelial-fibroblast co-culture system was exposed to TGF-β1 for 3 h at 37 °C. Then, (a, b) WST-1 reagent was added to the apical and basal regions. Cell viability was expressed relative to the control (% of Control). (c) The NET resistance value was measured before and after chemical treatment. The TEER value was calculated as (resistance) x cm² (surface area of the monolayer). The bar represents the difference in TEER value before and after treatment. (d) To determine reactive oxygen species of MRC-5 cells seeded on Basal side, the basal area was treated with DCF-DA reagent at 37 °C for 30 min and observed under a fluorescence microscope (x100). Oxidative stress was expressed compared to the control as fold induced. (e) After treatment for 3 h, the supernatant was collected and the levels (continued) of cytokines (IL-1α, IL-4, IL-6, and TNF-α) were measured. Cytokine level was expressed compared to controls as fold induction. Values are significantly different from controls. *p < 0.05, **p < 0.01.

In addition, TEER was measured to determine the integrity of tight junction dynamics in a cell culture model of epithelial monolayer [22]. As shown in Fig. 3c, the TEER value of TGF-β1 treated cells was significantly decreased compared to the control group, showing a higher TEER change compared to the TEER value before exposure.

The ROS production can be induced by oxidative stress, being linked to anti-oxidant mechanisms, inflammation, and apoptosis [23]. ROS generation in an MRC-5 cell in the basal area of the co-culture model was measured using the DCFDA assay at the end of exposure for 3 h and significantly increased approximately 11-fold compared to control group (Fig. 3d).

Expression of fibrosis-related secretory cytokines, namely IL-1α, IL-4, IL-6, and TNF-α was quantified using multiplex cytokine analysis in apical part (Fig. 3e). These secreted cytokines were also known to be related to inflammation [24], and among them, only IL-6 and TNF-α levels were increased approximately 2-fold by TGF-β1 exposure. On the other hand, IL-4 and IL-1α did not show significant changes due to TGF- β1 exposure.

Functional evaluation using epithelial-fibroblast co-culture system

Functional changes of hAECB cells exposed to TGF-β1 were confirmed through ciliated cell morphology and cilia beat frequency (CBF). As shown in Fig. 4a, the cilia of differentiated hAECB cells exposed to TGF-β1 showed a thinner shape compared to the control group.

Figure 4.

Functionality analysis using 3D Epithelial-fibroblast co-culture system after 3 h of exposure to TGF-β1. (a) Differentiated hAECB cells were stained with hematoxylin & eosin (H&E), and changes in the cilia were observed after exposure to TGF-β1. (b) The cilia beating area of differentiated epithelial cells under ALI conditions. The cilia beating area was visualized by distinguishing between an area where beating was not detected and an area where beating was detected. The cilia beating area is the percentage (%) of the area with movement relative to all pixels in the FOV, calculated as the mean and median (25th percentile to 75th percentile). (c) To observe MRC-5 cell migration, the cells were co-cultured with hAECB cells treated with TGF-β1 for 9 h. Cell migration was observed microscopically over time and expressed relative to control (fold induction). (d) Conditioned medium obtained from the basal region was collected and exposed to a collagen gel seeded with MRC-5 cells. Collagen contraction was observed over time using a microscope and expressed compared to control (magnification-derived). Values are significantly different from control at *p < 0.05, **p < 0.01.

Changes in cilia shape can lead to changes in their function. Therefore, CBF was measured to check cilia function. The cilia beating area at 9 FOVs between control and TGF-β1 is shown in Fig. 4b. CBF in hAECB cells exposed to TGF-β1 showed a significant decrease (**p < 0.01) of approximately 1.5-fold compared to control group. During fibrosis, TGF-β1, a major profibrotic growth factor, induces fibroblasts to differentiate into myofibroblasts [13] . When hAECB cells on apical area were treated with TGF-β1, MRC-5 cells seeded at the basal surface may be indirectly affected by TGF-β1. Therefore, cell migration and gel contractility, being key characteristics of myofibroblasts, were evaluated in MRC-5 cells on the basal surface. As a result, both cell migration (Fig. 4c) and gel contraction (Fig. 4d) showed a significant increase compared to control groups.

Discussion

Considering the limitations of animal models in predicting the safety of inhaled substances in humans, research efforts have been focused on developing human-mimic models [10]. Complex 3D airway models composed of epithelium and fibroblasts have been developed, but most of these studies are limited to identifying cellular damage and inflammation, and few studies have taken full advantage of the sophisticated configuration of these models [25, 26].

Interaction fibroblast and epithelial cell is very important in the induction of pulmonary fibrosis. Respiratory epithelial cells participate in the induction and regulation of immune responses to injury and inhaled insults through the secretion of proinflammatory cytokines [6, 27, 28]. These changes in epithelial cells induce the differentiation of fibroblast, leading to wound-healing responses in respiratory system, such inflammatory responses, fibroblast activation, and crosstalk with other cell populations [29]. To better reflect this concept, in this study, we designed a multi-analysis model that allows simultaneous assessment of toxicological and functional changes using a 3D Epithelial-fibroblast co-culture system, i.e., apical epithelial cells and basolateral fibroblasts.

In order to apply a multi-analysis model to evaluate pulmonary fibrotic responses, verification of integrity in 3D epithelial cells and fibroblast co-culture systems is very important and essential. In the apical bronchial epithelial cells, the TEER has been used to monitor the formation of tight junctions and evaluate the integrity of cell monolayers [19]. In our system, the TEER value of differentiated hAECB under ALI conditions remained stable at around 400 Ω.cm2 (Fig. 2a), consistent with the Leung’s results[30]. But, complete differentiation into mucociliary tissue under ALI conditions is depend on the ALI differentiated medium and may not completely match the TEER value [30]. Another report [31] suggested that TEER above 400 Ω.cm2 can reveal crucial phenotypic and functional characteristics of differentiated hAECB cells, in which ciliated cells and goblet cells are present. We confirmed the morphology of fully differentiated pseudostratified epithelial layer and ciliated epithelial cells using H&E staining (Fig. 2b) and also ICC staining identified goblet cell expressing MUC5AC (Fig. 2c). In addition, cilia motility was quantified for functional evaluation of ciliated epithelial cells and the proportion of cilia beating cells among all differentiated cells was approximately 60 % (56 % - 61 %) (Fig. 4b). According to Leung’s report [30], when hAECB cells were differentiated using the same culture medium (Pneumacult™, Epithelix) we used, the distribution of ciliated bronchial epithelial cells among the total differentiated cells was approximately 70 %, similar to our results. Based on these results, apical epithelial cell in the coculture model used in this study was judged to have well derived morphological and functional tissue forms. TGF-β1 is increased in Cystic Fibrosis airways, contributing to airway inflammation [32, 33], so it is considered a key mediator of pulmonary fibrosis [21]. In apical region, differentiated hAECB cells treated in TGF-β1 showed a significant decrease of TEER. TGF-β1 induces a decrease in TEER, which is known to be induced by damage to epithelial cell integrity, such as loss of tight junctions [34]. Another study also reported that TGF-β1 reduces lung epithelial barrier function and plays an active role in the pathophysiology of ALI [35, 36, 37]. Cilia and mucociliary clearance are the most important physiological defense mechanism of the airway epithelium [19, 38] Therefore, determining ciliary density through quantitative analysis of ciliary beating area can be a very important indicator for the integrity of airway epithelial cells [39]. When TGF-β1 was exposed in apical part of the co-culture system, the hAECB cells in apical side were observed histological changes and a reduction in cilia beating area were observed (Fig. 4a and 4b). This reduction in ciliary beat area in cystic fibrosis cells has been reported to be induced by TGF-β1 mediated airway dehydration [40].

On the other hands, respiratory epithelial cells participate in the induction and regulation of immune responses to injury and inhaled insults through the secretion of proinflammatory cytokines [6, 27, 28]. Epithelial cells-derived cytokine, TGF-β1 had a central role in the generation of the pulmonary immune response [41]. Apical solution exposed to TGF-β1 showed significant increase of IL-6 and TNF-α levels (Fig. 3e), which are important cytokines in the early inflammatory response [42]. IL-6 and TNF-α levels can be increased by TGF-β1 exposure [43]. It has been reported that TNF-α can induce a pro-fibrotic effects by acting directly on fibroblasts [44]. Secretion of these inflammatory cytokines in apical area has the potential to activates the function of basal side fibroblasts. In this study, MRC-5 cells in the basal region showed increased cell migration and contractility (Fig. 4), indicating that they were indirectly affected by hAECB cells in the apical region exposed to TGF-β1. Migration of myofibroblast to the site of injury is an essential factor in wound healing, which is known to play a role in fibrosis and wound contraction through increased matrix synthesis [45, 46]. Therefore, it is believed that the migration and contractility of fibroblast occurred as a response to damage of epithelial cell caused by TGF-β1. In particular, the increase in cell migration and contractility of fibroblasts is considered to be one of the fibrotic reactions.

To summarize, in this study, we performed functional assessments simultaneously in addition to cytotoxicity assessments and established a model that allows simultaneous analysis within a 3D epithelial-fibroblast co-culture system that closely resembles human anatomy. TGF-β1, known as a key disease mediator of IPF [20], was applied to the multi-analysis model and confirmed to induce a fibrotic response. This multi-analysis model suggests improved predictive accuracy and efficiency in toxicological evaluation, as an alternative to animal testing for the assessment of pulmonary fibrosis.

Acknowledgements

This work was supported by the Korea Environmental Industry & Technology Institute(KEITI) through Core Technology Development Project for Environmental Diseases Prevention and Management Program, funded by Korea Ministry of Environment(MOE) (RS-2021-KE00142) and through the Technology Development Project for Safety Management of Household Chemical Products Program funded by the Korea Ministry of Environment (MOE) (202300230430).

Notes

The authors declare that they have no conflict of interest.

CRediT author statement

MJK: Conceptualization, Methodology, Writing-Original draft preparation, HSH: Data curation, Writing-Original draft preparation, Formal analysis JHC: Software, Visualization, Investigation, ESY: Investigation, MIJ: Investigation, JHL: Supervision, SMO: Supervision, Project administration, Writing-Reviewing and Editing

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