<i>In vitro</i> study on the comparison of hypoxia-inducible factor-1α expression after particulate matter exposure in 2-dimensional and 3-dimensional cell culture models

Ji-O Ryu; Eunyoung Lee; Sang-Yun Lee; Hyeong Jun Cho; Jeong Uk Lim; Chang Dong Yeo; Yu-Jeong Seong; Seung Joon Kim; Dong Woo Lee

doi:10.51335/organoid.2025.5.e2

Organoid > Volume 5; 2025 > Article

Ryu, Lee, Lee, Cho, Lim, Yeo, Seong, Kim, and Lee: In vitro study on the comparison of hypoxia-inducible factor-1α expression after particulate matter exposure in 2-dimensional and 3-dimensional cell culture models

Original Article

Organoid 2025;5:e2.

Published online: February 25, 2025

DOI: https://doi.org/10.51335/organoid.2025.5.e2

In vitro study on the comparison of hypoxia-inducible factor-1α expression after particulate matter exposure in 2-dimensional and 3-dimensional cell culture models

Ji-O Ryu^1,^*

, Eunyoung Lee^2,^3,^*

, Sang-Yun Lee^4,^*

, Hyeong Jun Cho^2,³

, Jeong Uk Lim⁵

, Chang Dong Yeo⁶

, Yu-Jeong Seong¹

, Seung Joon Kim^2,³

, Dong Woo Lee¹

¹Department of Biomedical Engineering, Gachon University, Seongnam, Korea

²Postech-Catholic Biomedical Engineering Institute, Songeui Multiplex Hall, College of Medicine, The Catholic University of Korea, Seoul, Korea

³Division of Pulmonology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

⁴Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea

⁵MD Anderson Cancer Center Visiting Scholar in the Department of Investigational Cancer Therapeutics (Phase I), Houston, TX, USA

⁶Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Eunpyeong St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

Correspondence to: Dong Woo Lee, PhD Department of Biomedical Engineering, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam 13120, Korea E-mail: dw2010.lee@gmail.com

Seung Joon Kim, MD, PhD Division of Pulmonology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea E-mail: cmcksj@catholic.ac.kr

*These authors contributed equally to this work.

Received January 17, 2025 Revised February 16, 2025 Accepted February 19, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Particulate matter (PM) can penetrate deep into the lungs, leading to diseases through the induction of hypoxia and inflammation. The hypoxia-inducible factor-1α (HIF-1α) protein is well-established as a key factor in the hypoxic response, and PM exposure has been linked to hypoxia induction. To develop a model suitable for studying PM-induced hypoxia, we compared the expression of HIF-1α protein between a conventional 2-dimensional (2D) cell culture model and a 3-dimensional (3D) culture model.

Methods

BEAS-2B cells were cultured in 2D (using a 96-well plate) and 3D (utilizing a U-shaped pillar strip-based) models to compare cellular responses following PM treatment. Protein expression levels of HIF-1α were analyzed using Western blotting.

Results

The expression of HIF-1α increased in both 2D and 3D models in a time- and concentration-dependent manner following PM treatment, with a more pronounced and robust response observed in the 3D model. Furthermore, quantitative analysis using hypoxia dye staining confirmed that the 3D model exhibited a higher level of hypoxia induction after PM exposure compared to the 2D model.

Conclusion

In this study, HIF-1α expression in response to PM treatment was analyzed using both a 2D culture model and a U-shaped pillar-based 3D culture model. A more distinct, time- and concentration-dependent increase in HIF-1α expression was observed in the U-shaped pillar-based 3D model compared to the 2D model. These results highlight the suitability of the U-shaped pillar-based 3D model for investigating PM-induced hypoxia.

Keywords: Particulate matter; BEAS-2B; Hypoxic responses; Hypoxia-inducible factor 1-alpha; U-shape pillar

Introduction

Particulate matter (PM) is a major environmental pollutant that can penetrate deep into the lungs, inducing oxidative stress, inflammatory responses, and hypoxic reactions (Fig. 1A) [1]. This biological damage may contribute to the onset of various chronic diseases. In particular, hypoxic responses occur when tissues or cells are exposed to an environment with oxygen levels lower than normal [2,3]. Chronic or excessive hypoxic responses can lead to diseases, including tumors (i.e., cancer) (Fig. 1B) [4,5].

The hypoxic response is mediated by a key regulatory factor known as hypoxia-inducible factor (HIF), which is activated in response to oxygen levels (Fig. 1B). HIF-1α plays a pivotal role in this process by inducing various pathological changes [6,7]. PM activates HIF-1α through mechanisms closely linked to inflammatory and oxidative stress responses [5,8,9]. This activation promotes pathological tissue changes and can negatively impact the lungs, cardiovascular system, and nervous system [10].

The hypoxic response, along with inflammation and oxidative stress pathways induced by PM, affects not only the lungs but also vital organs such as the heart, brain, and liver [11,12]. In this process, HIF-1α activation may accelerate tissue damage and the progression of chronic diseases [13]. Understanding these mechanisms provides better insight into how environmental factors impact human health and serves as a foundation for developing effective prevention and treatment strategies.

Most existing studies on the relationship between PM and HIF-1α have used 2-dimensional (2D) cell culture systems [14]. However, 2D cell cultures have limitations in fully reflecting the physiological characteristics of actual tissues [15]. In 2D environments, cell-cell interactions and spatial structures are restricted, making it challenging to replicate the dynamic properties of the cellular microenvironment, such as signal transduction and substance diffusion. Conventional 2D cell culture studies fail to replicate physiological conditions, resulting in suboptimal HIF-1α activation. In contrast, 2-dimensional (3D) models can more accurately mimic cell-cell interactions and tissue structures [16]. By providing conditions closer to the physiological environment, 3D models enable a more precise investigation of intracellular signaling and substance transport [17,18]. Specifically, 3D models allow for a more accurate observation of HIF-1α expression and activation associated with hypoxic responses.

Therefore, this study aims to analyze the effects of PM on tissues using a 3D cell culture model, with a focus on the HIF-1α factor. It is expected that this approach will further elucidate the mechanisms of PM-induced HIF-1α activation.

Materials and Methods

Ethics statement: This study constituted a comprehensive analysis of previously released studies and thus, was not subject to the approval of the institutional review board.

1. Cell culture

The BEAS-2B cell line, derived from normal human bronchial epithelium (Fig. 2A), was cultured in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin to prepare a complete medium. In the 2D model, 5,000 cells per well were seeded into a 96-well plate containing 80 µL of medium and cultured in a 37°C incubator (Fig. 2B and 2C). For the 3D model, a U-shaped pillar strip was employed (Fig. 2E). BEAS-2B cells for the 3D model were mixed with Matrigel (50% v/v) at a concentration corresponding to 5,000 cells per 3 µL. The mixture was dispensed (3 µL per spot) onto the surface of each U-shaped pillar using a pipette, followed by gelation in a 37°C incubator for 1 hour to form the 3D model (Fig. 2F and 2G). After gelation, the U-shaped pillar strip with the 3D model was combined with a 384-well plate containing 80 µL of fresh medium [19] (Fig. 2H). Using these 2D and 3D models, the correlation between PM treatment and HIF-1α protein expression was analyzed under varying PM concentrations and time-dependent conditions.

2. PM treatment

PM (cat. no. 1648a; National Institute of Standards and Technology) was prepared as a 40 mg/mL stock solution. For experiments, it was diluted with culture medium to final concentrations of 0, 25, 50, and 100 µg/mL. In the 2D model, the PM-supplemented medium was directly added to the wells containing cells (Fig. 2D). In the 3D model, the PM-containing medium was added to a 384-well plate, and the pillar strips with cultured cells were inserted for further incubation (Fig. 2I).

3. Western blotting

To harvest cells from the 2D model, wells were washed with phosphate-buffered saline, followed by the addition of 50 µL of TrypLE to each well and a 5-minute incubation. For the 3D model, 80 µL of TrypLE was added to each well, and the cells were incubated for 25 minutes to facilitate retrieval. Protein analysis was performed using Western blotting, with 20 µg of protein from each sample prepared in 20 µL and loaded onto the gel. Electrophoresis was conducted at 100 V for 1 hour and 15 minutes, and proteins were subsequently transferred to a membrane at 350 mA for 1 hour. For HIF-1α detection, primary antibodies Rabbit anti-HIF-1α (cat. no. 14179S; Cell Signaling Technology) at a 1:1,000 dilution and Mouse anti-β-actin (cat. no. sc-47778; Santa Cruz) at a 1:5,000 dilution were used. Secondary antibodies Goat anti-mouse HRP (cat. no. ab6789; Abcam) at a 1:5000 dilution and Goat anti-rabbit HRP (cat. no. ab6721; Abcam) were employed to detect protein expression (Fig. 2J).

Results

1. Cell viability and hypoxia induction in 2D and 3D models following PM treatment

After treating BEAS-2B cells with varying concentrations of PM, cell viability was assessed. The results indicated that cell viability decreased in both 2D and 3D models as the PM concentration increased, with a more pronounced decline observed in the 2D model. This suggests that the 2D model is more sensitive to lower PM concentrations than the 3D model in studies of PM treatment (Fig. 3). When experiments are conducted using 2D models at the same PM concentration as the 3D model, the limited number of cells in the 2D model may not provide optimal conditions. Furthermore, because the effective PM concentration in the 2D model appears lower than in the 3D model, physiological effects such as hypoxia may be less pronounced. Additionally, performing experiments at low concentrations makes it challenging to adequately capture concentration-dependent responses.

Hypoxia induction in the 2D and 3D models at the same PM concentration was evaluated using hypoxia dye staining. The analysis confirmed that the hypoxic condition in the 3D model was higher than in the 2D model upon PM treatment (Fig. 4A). Specifically, the fluorescence intensity in the 2D model at 50 µg/mL PM increased 3.9-fold compared to 0 µg/mL PM, whereas in the 3D model, the fluorescence intensity at 50 µg/mL PM was 290.1-fold higher when compared to the 2D model at 0 µg/mL PM. Additionally, within the 3D model, the fluorescence intensity at 50 µg/mL PM was 5.6-fold higher than at 0 µg/mL PM, confirming that PM treatment induced a stronger hypoxic response in the 3D model than in the 2D model (Fig. 4B).

2. Time-dependent expression of HIF following PM treatment

Following treatment with 50 µg/mL PM, HIF-1α expression was analyzed by Western blot at 0, 24, 48, and 120 hours in both the 2D and 3D models. The results showed a time-dependent increase in HIF-1α expression in both models (Fig. 5A and 5B). Notably, in the 3D model, HIF-1α expression increased sharply at the 120-hour time point, an effect more pronounced than in the 2D model (Fig. 5C).

These findings suggest that the 3D model more accurately reflects the physiological effects of PM. The 3D model’s complex microenvironment, including extracellular matrix (ECM) and cell-cell interactions, better captures the stress responses induced by PM treatment. Furthermore, the continuous increase in HIF-1α expression over time indicates that PM activates the cellular hypoxic response pathway, prompting cells to adapt to oxygen-deficient conditions.

The marked increase in HIF-1α expression at 120 hours in the 3D model highlights the enhanced cellular response to PM treatment and provides more physiologically relevant data.

3. Expression of HIF-1α following PM treatment at different concentrations

After treating BEAS-2B cells with PM at concentrations of 0, 25, 50, and 100 µg/mL for 24 hours, HIF-1α expression was analyzed by Western blot in both the 2D and 3D models. The results indicated a concentration-dependent increase in HIF-1α expression in both models (Fig. 6A and 6B). Notably, in the 3D model, HIF-1α expression increased sharply at 100 µg/mL, exhibiting a more pronounced effect compared to the 2D model (Fig. 6C).

These findings suggest that the 3D model more effectively simulates the physiological impact of PM. Its complex microenvironment, which includes ECM and cell-cell interactions [20], more accurately reflects the stress responses induced by PM treatment. Moreover, the sustained increase in HIF-1α expression indicates that PM activates the intracellular hypoxic response pathway, prompting cells to adapt to oxygen-deficient conditions.

Specifically, the rapid increase in HIF-1α expression at 100 µg/mL in the 3D model is significant, as it demonstrates that a stronger hypoxic response to PM treatment is induced in the 3D environment, thereby providing more meaningful physiological data. In addition, it was confirmed that hypoxic conditions were more effectively induced in the 3D model with U-shaped pillar strips. The effect on vascular endothelial growth factor (VEGF) expression is presented in Fig. S1. These findings demonstrate the potential of using the 3D model with U-shaped pillar strips for studying various hypoxia-related mechanisms, such as VEGF induction by PM treatment.

Discussion

PM is a major environmental toxicant that induces hypoxic responses by activating intracellular oxidative stress and inflammatory pathways, thereby increasing HIF-1α expression [5,8,9]. This study aimed to evaluate the physiological impact of PM on human tissues by comparing HIF-1α activation and cellular responses in 2D and 3D culture models.

The experimental results demonstrated that PM treatment significantly increased HIF-1α expression in a time- and concentration-dependent manner, indicating activation of hypoxic response pathways in cells. Notably, the hypoxic response induced by PM was more pronounced in the 3D model than in the 2D model, likely due to the complex microenvironment—including ECM and cell-cell interactions—present in the 3D model. Specifically, after 120 hours or under high-concentration PM treatment (100 µg/mL), the increase in HIF-1α expression in the 3D model was markedly higher than in the 2D model.

These findings underscore that the 3D model provides a more effective platform for investigating physiologically relevant responses, such as PM-induced HIF-1α activation.

In conclusion, the 3D culture model is an effective tool for evaluating the physiological effects of PM exposure and holds promise for advancing environmental toxicity assessments and the development of therapeutic strategies.

Supplementary Information

Supplementary materials are presented online (available at https://doi.org/10.51335/organoid.2025.5.e2).

Fig. S1.

Changes in hypoxia levels following exposure to normoxic or hypoxic conditions in 2-dimensional (2D) and 3-dimensional (3D) models formed using the A549 lung cancer cell line. (A) Hypoxia levels were assessed in 2D and 3D models using a hypoxia staining dye, with nuclei counterstained with Hoechst 33342. (B) The hypoxia level in the 3D model was 1.86-fold higher than that in the 2D model. (C) The expression of vascular endothelial growth factor (VEGF), a hypoxia-related marker protein, was analyzed in both models. (D) VEGF protein expression was 2.14-fold higher in the proposed 3D model under hypoxic conditions for 24 hours than in the 2D model under normoxic conditions.

organoid-2025-5-e2-Supplementary-Fig-1.pdf

NOTES

Conflict of interest

Seung Joon Kim has been an editors-in-chief of Organoid since 2021. However, Seung Joon Kim was not involved in the review process of this manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2020R1I1A306655012). This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (No. RS-2024-00453887). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00212410). This research was supported by a grant of Korean ARPA-H Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2024-00512449).

Authors’ contributions

Conceptualization: EL, SYL, HJC, JUL, CDY, SJK, DWL; Data curation: JOR, EL, SYL, HJC, JUL, CDY, YJS, SJK, DWL; Formal analysis: JOR, EL, SYL, HJC, JUL, CDY, YJS, SJK, DWL; Funding acquisition: SYL; Investigation: JOR, EL, HJC, JUL, CDY, SJK; Visualization: JOR, EL, SYL, YJS; Writing-original draft: JOR, EL; Writing-review & editing: all authors.

Data availability

Please reach out to the corresponding author to inquire about the availability of data.

Fig. 1.

Schematic of hypoxia and hypoxia-inducible factor 1-alpha (HIF-1α) changes induced by particulate matter (PM). (A) In normal lungs, PM exposure induces oxidative stress, inflammatory responses, and hypoxic reactions. (B) As the condition becomes chronic, the risk of cancer development increases, with HIF-1α regulating hypoxic responses. Illustrated by the authors and is created via BioRender with credit.

Fig. 2.

Two-dimensional (2D) and three-dimensional (3D) cell culture methods and particulate matter (PM) treatment workflow. (A) Prepare BEAS-2B cells and adjust the cell count to the appropriate concentration. (B) For 2D cell culture, seed cells onto a 96-well plate. (C) Culture the cells in a 37°C, 5% CO₂ incubator. (D) Treat the cultured cells with PM at the desired concentration. (E) For 3D cell culture, utilize U-shaped pillars to facilitate 3D structure formation. (F) Load the cells onto the U-shaped pillars. (G) Allow gelation in the incubator for 1 hour. (H) After gelation, continue culturing the cells in a 37°C, 5% CO₂ incubator. (I) Add PM-treated media into a 384-well plate and insert the U-shaped pillars to culture the cells. (J) Evaluate hypoxia-inducible factor 1-alpha (HIF-1α) expression levels using protein immunoblotting. Illustrated by the authors and is created via BioRender with credit.

Fig. 3.

Cell viability measurement following treatment with different particulate matter (PM) concentrations. (A) In both 2-dimensional (2D) and 3-dimensional (3D) models, cell viability decreased as PM concentration increased. (B) Representative images of cell viability following treatment with various PM concentrations. Statistical differences in cell viability are indicated relative to each control group (2D or 3D PM 0 μg/mL) and the corresponding experimental groups (2D or 3D PM 25-400 μg/mL). ^***p<0.001.

Fig. 4.

Changes of Hypoxia Conditions in 2D and 3D Models Following PM Treatment for 120 Hours Using Hypoxia Staining Dye Analysis (A) 2D and 3D models were stained with nucleus and hypoxia staining dye. (B) The 3D model was shown quantitatively to induce 209.1 times more hypoxic conditions than the conventional 2D model. Statistical significance of Hypoxia dye intensity between PM-treated and non-treated conditions in 2D and 3D models was indicated as ^* for p-value >0.05 and ^** for p-value >0.01.

Fig. 5.

Changes in hypoxia-inducible factor 1-alpha (HIF-1α) expression following particulate matter (PM) treatment at various time points. (A) Cell images captured at different time points following PM treatment in both 2-dimensional (2D) and 3-dimensional (3D) models. (B) Western blot results demonstrated that HIF-1α expression increased over time in both models. (C) Quantitative analysis confirmed a time-dependent increase in HIF-1α expression, with a notably sharp increase at 120 hours in the 3D model. The statistical significance of HIF1A protein expression over time following PM 50 µg/mL exposure in 2D and 3D models was indicated as ^* for p-value >0.05.

Fig. 6.

Changes in hypoxia-inducible factor 1-alpha (HIF-1α) expression following treatment with various particulate matter (PM) concentrations. (A) Cell images after treatment with different PM concentrations in both 2-dimensional (2D) and 3-dimensional (3D) models. (B) Western blot results showed that HIF-1α expression increased with rising PM concentrations in both models. (C) Quantitative analysis confirmed a concentration-dependent increase in HIF-1α expression, with a marked increase observed at 100 μg/mL in the 3D model. The statistical significance of HIF1A protein expression according to PM exposure concentrations (0, 25, 50, and 100 µg/mL) in 2D and 3D models was indicated as ^* for p-value >0.05.

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