Respiratory microphysiological system in respiratory research: recent advances and future prospects

Jiaxiang Yin; Zirong Bi; Tiankai Dai; Xiaoyue Zhu; Tao Xu; Huisheng Liu

doi:10.1186/s12931-025-03327-1

. 2025 Jul 26;26:253. doi: 10.1186/s12931-025-03327-1

Respiratory microphysiological system in respiratory research: recent advances and future prospects

Jiaxiang Yin ^1,², Zirong Bi ¹, Tiankai Dai ^1,³, Xiaoyue Zhu ^1,³, Tao Xu ^1,^2,^✉, Huisheng Liu ^1,^2,^✉

PMCID: PMC12296607 PMID: 40713673

Abstract

Respiratory diseases encompass a broad range of conditions that affect the airways, alveolus, and other structures involved in breathing. With respiratory viruses emerging and spreading rapidly, and factors like air pollution, smoking, occupational hazards, and genetics as major risk contributors, respiratory diseases have emerged as a primary cause of illness and mortality around the globe. The development of in vitro respiratory microphysiological systems (MPS), like lung organoids and lung-on-a-chip models, is essential for preventing and treating these diseases effectively. In this review, we introduce the generation of lung organoids from different cellular sources and their applications in modeling lung development, disease models, and drug screening. Then, we discuss the establishment of various types of lung-on-a-chip models and their applications in disease modeling and drug screening. Finally, we outline the challenges confronting respiratory MPS and provide an outlook on their future perspectives.

Keywords: Respiratory microphysiological system, Lung organoids, Lung-on-a-chip, Disease models, Drug screening

Introduction

Respiratory diseases embodiment of a significant worldwide danger to human health, and impose a heavy burden on healthcare systems. With societal development, increasing population pressures, and worsening environmental pollution, the occurrence and death rates of conditions like chronic obstructive pulmonary disease (COPD), asthma, lung cancer, and lung infections are rising both domestically and internationally. Reports indicate that chronic lung diseases cause over 3 million deaths annually, putting them in the third spot for the most common cause of death globally [1–3]. At present, the advancement of therapies for respiratory diseases is progressing slowly, primarily due to the absence of relevant human models that can reliably replicate the onset and progression of these diseases [4]. This limitation hinders advancements in the discovery of prevention and therapy strategies. Therefore, there is a critical need to create new types of in vitro respiratory microphysiological system (MPS) models. These models would provide a deeper knowledge of the mechanisms behind lung infections, disease progression, host responses, and cellular repair processes, ultimately aiding in the advancement of novel targeted therapies for lung diseases.

The lung is an intricate organ that consists of around 58 different types of cells [5]. Its structure includes a proximal tubular branching system, made up of the bronchioles, which connect to individual airway ducts. As the bronchioles branch and become smaller, they lead to even narrower tubes known as alveolar ducts, which eventually connect to the alveolar regions where gas exchange takes place [6]. Lung development involves a series of complex processes, mainly including 6 stages (endodermal induction, anterior-posterior and dorsal-ventral patterning, lung-specific regionalization, lung budding, branching morphogenesis, and eventual maturation). Consequently, constructing a lung organ model is a significant challenge due to each cell type serves a distinct function in specific regions of the lung. However, with the rapid advancements in organoid and organ-on-a-chip (OOC) technologies, it has become increasingly feasible to create MPS that simulate human organs or the specific functions of organs.

Cell cluster, spheroid, and organoid are commonly used 3D cell models in biomedical research, but they exhibit significant differences in structure, function, and application (Table 1). Cell clusters are simple 3D structures formed by spontaneous or artificial aggregation of cells, typically lacking a defined organizational architecture. Their characteristic feature is that cells are loosely bound via adhesion molecules (e.g., E-cadherin) and may consist of a single or multiple cell types, but they show no functional differentiation or polarity. They are often used in drug screening or preliminary studies of cell-cell interactions. Spheroids are spherical 3D cell aggregates induced through suspension culture or low-adhesion substrates, partially mimicking the features of tumors or tissues. Their defining characteristic is a core-periphery structure (e.g., a hypoxic core and a proliferatively active outer layer), resembling the microenvironment of solid tumors in vivo. They may be formed from tumor cells, stem cells, or primary cells, but lack organ-specific structures. Spheroids are commonly used in cancer research, drug permeability testing, and radiation therapy response experiments. Organoids are miniature organ-like structures formed through the self-organization and differentiation of stem cells (pluripotent stem cells or adult stem cells), exhibiting organ-specific cell types and functions. Their defining features include containing multiple cell types and recapitulating partial organ structure and function (e.g., crypt-villus structures in intestinal organoids, neuronal layering in brain organoids). They rely on growth factors and extracellular matrices (e.g., Matrigel) for microenvironmental support. Organoids are widely used in disease modeling, regenerative medicine, and personalized medicine. The concept of 3D organotypic culture dates back to the 1920s, when small pieces of epithelial tissue or dissociated cells were cultured in purified 3D extracellular matrix (ECM) gels, resulting in the formation of organ-like structures [7–10]. In 2009, a major advancement in organoid technology was made when Clevers and his team demonstrated that Lgr5⁺ stem cells could grow and self-organize without the need for a non-epithelial cell niche [11]. This discovery paved the way for the establishment of various organoids, including gastric organoid [12], optic-cup organoid [13], intestinal organoid [14], thyroid organoid [15], pancreas organoid [16], liver organoid [17], cerebral organoid [18], salivary organoid [19], prostate organoid [20], lung organoid [21], gastric organoid [22], mammary gland organoid [23], kidney organoid [24], placenta organoid [25], skin organoid [26], etc. Organoids were recognized for their extraordinary capacity as resources for studying human biology and diseases, earning them the title of Method of the Year by Nature Methods in 2017 [27].

Table 1.

The key differences among cell cluster, spheroid and organoid

	Cell Cluster	Spheroid	Organoid
Complexity	Low (simple aggregation)	Medium (microenvironment mimic)	High (organ-specific architecture)
Cell Source	Any cell type	Tumor cells/stem cells/primary cells	Stem cells (pluripotent or adult)
Self-organization	Weak	Moderate	Strong (self-patterning & differentiation)
Functionality	No specific function	Partial pathological/physiological function	Near-native organ function
Culture Conditions	Conventional suspension culture	Low-adhesion substrate/serum-free medium	Growth factors + ECM (e.g., Matrigel)
Main Applications	Preliminary cell interaction studies	Tumor microenvironment, drug screening	Disease modeling, regenerative therapy

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OOC is a synthetic model that combines a microfluidic chip with living cells to mimic the architecture, physiological functions, and biomechanics of human organs [28–30]. The OOC technology surfaced in 1990s, and shortly after, Shuler’s lab introduced the ides of “human-body-on-a-chip” as a means to study human physiology [31]. Benefiting from the advantages of microfluidic chips, such as miniaturization, automation, integrability, compact size, rapid response, flexible structural design, precise control over fluids and physical/chemical factors [32–35], OOC technology has enabled the creation of near-physiological microenvironments [36]. This has been achieved through the integration of microelectronics, materials science, chemistry, biology, and engineering.

In this review, we offer a summary of the latest advancements in respiratory MPS, focusing on lung organoids and lung-on-a-chip models. We start with a comprehensive introduction of lung organoid and its application. Next, we describe the lung-on-a-chip respiratory MPS, highlighting its role in disease modeling and drug discovery. Finally, we discuss the current challenges and future perspectives of respiratory MPS in both industrial and clinical applications.

Lung organoids and its application

Culture techniques

Conventional 2D cell culture refers to cultivating cells on a culture dish or plate, and has been widely used in the field of life sciences for many years. This technique is favored for easy operation, cost-effectiveness, and high-throughput capabilities. However, 2D culture method is not capable of replicating the complex 3D environment in which cells naturally grow in the body, leading to significant differences in cell morphology and behavior compared to in vivo conditions. In turn, the findings from 2D cell culture experiments often do not align with animal or clinical studies [37]. To address this limitation, researchers have spent the past three decades developing in vitro 3D culture systems that better replicate physiological conditions.

In the 1990s, publications on 3D cell culture models were limited to around 10 per year. However, In the past 20 to 30 years, the field has undergone rapid development, with the volume of publications surpassing 3,500 in 2024. 3D cell culture techniques mainly include the following 7 types [37]. The first type is the hanging drop method (Fig. 1A) [38]. The method entails placing a droplet of cell suspension underside of the lid of a culture dish, where cells cling to the lid because of surface tension. The lid is then positioned on a culture dish containing PBS. Eventually, cells aggregate at the gas-liquid interface at the tip of the droplet and spontaneously form spheroids [37]. The second method is spontaneous spheroid formation (Fig. 1B) [39]. This method involves using non-adherent surfaces or ultra-low attachment plates to prevent cell adhesion, thereby causing the cells to cluster together and form spheroids. The third method is suspension culture (Fig. 1C) [40, 41]. This method involves stirring or increasing medium viscosity to allow cells to grow in suspension in vessels (such as spinner flasks and bioreactors). The fourth method is based on hydrogel scaffold models (Fig. 1D) [42]. This method involves seeding cells onto solidified hydrogels or mixing cells with liquid hydrogels before solidification, allowing cells to grow in the 3D ECM microenvironment provided by the hydrogel. The fifth method is magnetic levitation (Fig. 1E) [43]. In this method, magnetic iron oxide nanoparticles are co-incubated with cells overnight, and the treated cells are then transferred into an ultra-low attachment plate. A neodymium magnet is promptly positioned above the lid, and spheroids begin to form at the gas-liquid interface within a few hours due to the levitation effect of the magnet. The sixth method is bioprinting (Fig. 1F) [44]. This method involves constructing complex structures and physiological microenvironments mimicking those inside the body by using different bio-inks (biomaterials, growth factors, and cells) in vitro. The seventh method is microfluidics (Fig. 1G) [45, 46]. Microfluidic systems can culture and continuously inject live cells in micrometer-sized chambers, allowing precise control of the cell microenvironment, input of growth factors and nutrients, and removal of metabolites.

Fig. 1 — 3D cell culture methods used for generating spheroids. (A) Handing drop methods. (B) Spontaneous spheroid formation on the ultra-low attachment plates. (C) Suspension cultures. (D) Scaffold-based models. (E) Magnetic levitation. (F) 3D Bioprinting methods. (G) Microfluidics techniques

Organoids formation

The formation of organoids is informed by the study of in vivo organogenesis [47]. Organogenesis refers to the process of organ formation in developing embryos. Pluripotent stem cells (PSCs) can differentiate into a variety of cell types, making it essential to guide their differentiate into ectoderm mesoderm or endoderm in vitro. Once the germ layers are established, specific signals involved in organ formation can be regulated to drive the generation of corresponding organoids [48–50]. This process is facilitated by providing the necessary cellular factors and recreating the appropriate microenvironment, guided by principles of engineering design, synthetic biology, and systems biology.

Taking the generation of lung organoids in vitro as an example of mimicking in vivo lung organogenesis, the developmental process of the lung in vivo involves several key stages. These include gastrulation (germ layer induction), formation of the gut tube (anterior patterning), development of the lung bud (tissue specification), branched morphogenesis, and the maturation of lung tissue [51]. In vitro, PSCs are first induced to form definitive endoderm (germ layer induction). They are then directed toward the anterior foregut endoderm (anterior patterning) by inhibiting BMP and TGF-β [52]. Lung progenitor cells (LPCs) (tissue specification)—also known as “ventralized” anterior foregut endoderm (VAFE)—is primarily driven by signaling pathways involving WNT, BMP, retinoic acid, and FGF [21, 52]. This process leads to the activation of NKX2-1 expression, which is a key marker of lung epithelial lineage. The final stage is maturation, involving the differentiation of LPCs into airway and alveolar lineages, which then further develop into airway and alveolar organoids, respectively.

Lung organoids

The initial cell population is vital to the formation of any organoid, as it directly impacts the variability, heterogeneity, and functional properties of the resulting structures. For lung organoids specifically, the cell sources mainly include 3 types (Table 2).

Table 2.

Lung organoids derived from different cell sources

Cell Sources		Types of Lung Organoids	References
PSCs	induced PSCs	airway and alveolar structures	[53]
		alveolar organoids	[54]
		alveolar organoids	[55]
		airway organoids	[56]
		airway and alveolar organoids	[57]
	embryonic stem cells	airway spheroids	[58]
	embryonic stem cells	lung organoids	[59]
patient-derived samples	tumor tissue of primary lung cancer patients and patient-derived xenograft	NSCLC organoids	[60]
	patient-derived tissues	LADC organoids	[61]
	fragments tumor tissues	LCOs	[62]
	nasal brushing samples of PCD patients	COPD bronchial organoids	[63]
	MSE samples	LCOs	[64]
normal samples	bronchial epithelial cells, fibroblasts, and microvascular endothelial cells	airway organoids	[65]
	primary lung tissues	alveolar organoids	[66]
	primary lung tissues	airway and alveolar organoids	[67]

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PSCs: pluripotent stem cells; NSCLC: non-small cell lung cancer; LADC: lung adenocarcinoma; LCOs: lung cancer organoids; PCD: primary ciliary dyskinesia; COPD: chronic obstructive pulmonary disease; MSE: malignant serous effusion

Pluripotent stem cell-derived lung organoids

PSCs, whether induced or embryonic, possess the unique capacity for self-renewal and differentiation into any cell type, making them an appealing choice for generating organoids. Induced PSCs (iPSCs) technology involves reprogramming fully differentiated somatic cells into PSCs by introducing specific transcription factors. This groundbreaking technique was first reported by Shinya Yamanaka of Kyoto University in 2007 [68]. Embryonic stem cells (ESCs) are obtained from primitive gonads or early embryos. These cells have remarkable features, including indefinitely proliferation, and the special potential for differentiation into all cell types [69].

In 2011, Snoeck et al. was the first to achieve success in differentiate human iPSCs (hiPSCs) and human ESCs (hESCs) into lung epithelial cells [52]. By 2012, the stepwise differentiation of mouse PSCs was demonstrated through the generation of pluripotent NKX2-1⁺ embryonic LPCs [70, 71]. In 2017, they reported the derivation of LPCs from human PSCs (hPSCs), with 51% of the cells expressing NKX2-1 [53]. These LPCs subsequently formed lung bud organoids (LBOs). After xenotransplantation and 3D culture in Matrigel, the LBOs formed early airway and alveolar structures (Fig. 2A). However, the differentiation protocols produced a mix of cell types, leading to inconsistencies in the composition and development of lung organoids.

Fig. 2 — Differentiation protocols for PSCs-derived lung organoids. (A) A 2D differentiation method for generating lung epithelial cells (i) and a protocol illustrating the generation lung bud organoids (ii). Reproduced with permission from Ref [53]., Copyright 2017, Springer Nature. (B) A stepwise differentiation for generating proximal airway organoids. Reproduced with permission from Ref [58]., Copyright 2016, Cell. (C) A protocol for deriving alveolar organoids from iPSCs. Reproduced with permission from Ref [54]., Copyright 2017, Springer Nature. (D) An alveolosphere differentiation protocol from human PSCs. Reproduced with permission from Ref [55]., Copyright 2019, Springer Nature. (E) An airway-directed differentiation protocol from human PSCs. Reproduced with permission from Ref [56]., Copyright 2021, Cell. (F) An airway and alveolar organoids differentiation protocol from iPSC-derived NKX2-1 + lung bud tip progenitor organoids. Reproduced with permission from Ref [57]., Copyright 2022, Biologists

Based on a stepwise differentiation study of alveolar epithelial progenitor cells (AEPCs), Mishima et al. identified carboxypeptidase M (CPM) [72]. They suggested that CPM is a surface marker for NKX2-1⁺ VAFE cells in both human and mouse fetal lungs. Utilizing SFTPC-GFP reporter hESCs and a fetal human lung fibroblast 3D co-culture system, they demonstrated that the CPM⁺ cells could form AEPCs, further confirming that CPM is a marker of VAFE cells. Subsequently, they developed a differentiation method to generate airway spheroids from CPM⁺ VAFE cells (Fig. 2B) [58]. Relying on CPM and NKX2-1 as markers, they devised an efficient method for producing and expanding alveolar organoids containing SFTPC⁺ alveolar progenitor cells derived from hiPSCs (Fig. 2C) [54]. These SFTPC⁺ cells exhibited self-renew capacity, transcriptomic and morphological features akin to alveolar epithelial type II (AT2) cells, and the ability to differentiate into alveolar epithelial type I (AT1) cells. Using gene-editing technology, Kotton et al. engineered NKX2-1^GFP reporter hPSCs, facilitating obtainment of NKX2-1-expressing LPCs in vitro [73]. Following purification by flow cytometry, these LPCs exhibited evidence of lung epithelial maturation. Remarkably, even without mesenchymal co-culture, NKX2-1⁺ populations were capable of forming epithelial-only spheroids in 3D culture. By optimizing growth factors and small molecules and morphogenic conditions inspired by lung development, diverse lung organoids and cell types-such as alveolar organoids (Fig. 2D) and airway organoids (Fig. 2E)-were derived from NKX2-1^GFP reporter hPSCs [55, 56, 74, 75]. This differentiation approach yields LPCs with higher NKX2-1 expression purity. However, the complexity of sorting procedures can reduce cell viability and increase the risk of contamination. In addition, the differentiation process, which transitions hPSCs to LPCs in a 2D culture format, differs significantly from the 3D microenvironment characteristic of lung development in vivo.

Using 3D culture technology, Spence et al. manipulated developmental signaling pathways by adding growth factors to generate floating VAFE spheroids from hPSCs [76]. These spheroids were subsequently encapsulated in Matrigel for continued culture, leading to the formation of human lung organoids (HLOs). The resulting HLOs exhibited structural features reminiscent of the native lung, including airway-like epithelium and alveolar-like regions populated by appropriate cell types. By optimizing lung organoids differentiation protocols and utilizing flow cytometry, Spence et al. isolated NKX2-1⁺/CPM⁺ cells and expanded them into bud tip organoids (BTOs), maintaining approximately 80% NKX2-1⁺/CPM⁺ cells [57]. These BTOs were further formed airway or alveolar cell types, producing organoids with organized airway or alveolar epithelia (Fig. 2F). Despite these advances, the differentiation protocols face challenges, including inconsistent yield and variable quality of anterior foregut endoderm (AFE) spheroids.

To enable large-scale production of 3D lung organoids, Gomperts et al. developed a detailed bioengineering protocol for generating lung organoids [77]. Using alginate hydrogel microbeads as a biomimetic scaffold functionalized with collagen I/dopamine coating, the team co-cultured fibroblasts, epithelial cells, and other cell types in a rotating bioreactor to form organoids with distal lung architecture and multicellular composition. This method is modular and scalable, making it suitable for personalized disease modeling (e.g., pulmonary fibrosis) and drug screening.

Patient-derived lung cancer organoids

Patient-derived lung cancer organoids (LCOs) refer to 3D spherical structures generated from tissues obtained from lung cancer patients [78]. These organoids are composed of various types of cells and exhibit organized growth patterns. By processing tissue samples from diverse patients and culturing them under specific conditions, researchers have successfully developed several lung cancer organoid models [79, 80].

Non-small cell lung cancer (NSCLC) is the most frequent lung cancer subtype, making up around 84% of all cases, and has a 5-year survival rate of merely 15% [81]. Tsao et al. developed NSCLC organoids by isolating tumor tissue of primary lung cancer patients and patient-derived xenograft (PDX) into single cells and culturing them in a Matrigel-based system (Fig. 3A) [60]. These organoids faithfully retained the histological features, tumorigenicity, and therapy responsiveness of their corresponding patient and PDX tumor tissues. The establishment success rate for LCOs from primary tumor samples was 88% (57/65). Lung adenocarcinoma (LADC), a subtype of NSCLC, has also been successfully modeled using patient-derived tissues. Huang et al. proposed a detailed method to form LADC organoids (Fig. 3B) [61]. This method achieved a success rate of approximately 80%, resulting in obtaining 12 patient-derived LADC organoids [82]. Comprehensive genomic and histopathological analyses revealed that these organoids recapitulated the tissue architecture, genomic landscape and gene expression features of the original patient tumor tissue.

Fig. 3 — Protocols for generation of patient-derived LCOs. (A) A protocol for establishing NSCLC organoid from tumor tissues. Reproduced with permission from Ref [60]., Copyright 2020, American Association for Cancer Research. (B) A protocol for generating LADC organoids from clinical patient tissue samples. Reproduced with permission from Ref [61]., Copyright 2021, Elsevier. (C) A workflow for generating LCOs using mechanically processed patient tumor tissues. Reproduced with permission from Ref [62]., Copyright 2021, Springer Nature. (D) Procedure for establishing COPD organoids. Reproduced with permission from Ref [63]., Copyright 2021, EMBO Press

Small cell lung cancer (SCLC) represents about 15% of all lung cancer cases and has a five-year survival rate of under 5%. Compared to NSCLC, SCLC has the features of a rapid doubling time, high malignancy, and a propensity for early and extensive metastasis [83]. Jeong et al. successfully developed a protocol to generate SCLC organoids by digesting tumor biopsy samples into single cells and culturing them in a Matrigel-based system [84]. In addition, experimental results showed that adding WNT3A or R-spondin1 enables long-term expansion of the generated SCLC organoids, while preserving gene characteristics, molecular features, and morphological structures of their parental tumors throughout and after the expansion process. Most organoids, including those mentioned above, are created by enzymatically digesting tumor tissues into single cells, embedding them in Matrigel, and culturing them. Alternatively, mechanical processing can be used to generate tumor organoids. Liu et al. developed a mechanical sample processing method that fragments tumor tissues, enabling the generation of over 100 LCOs within just three days (Fig. 3C) [62]. This method achieved a success rate of 79%, with organoids established from 55 out of 71 LADC, 18 out of 23 squamous cell carcinomas, 4 out of 4 SCLC cases, and 4 out of 5 other lung cancer samples. LCOs generated using this method preserve the genetic and histological features of their parent tumors. To investigate the mechanisms of cellular interactions and tumor recurrence in the tumor microenvironment after chemotherapy, Gomperts et al. developed a 3D SCLC organoid co-culture model based on an alginate microbead scaffold [85]. By co-culturing SCLC cell lines (e.g., H526) with primary adult lung fibroblasts (ALFs) at a 4:1 ratio on functionalized microbeads, the model successfully recapitulated key features of SCLC, including rapid proliferation, invasive growth, and expression of classic neuroendocrine markers (e.g., CGRP, Chromogranin A), closely mimicking the pathological characteristics of patient tumors and cell line-derived xenograft (CDX) models.

Cystic fibrosis (CF) is a prevalent genetic condition marked by the buildup of thick mucus in the ducts and airways of several organs, especially the lungs and pancreas. This causes symptoms like breathing difficulties, chronic coughing, recurring respiratory infections, pancreatic enzyme deficiency, digestive issues, malnutrition, and stunted growth. The condition usually presents in infancy or early childhood. Clevers et al. reported a method for establishing long-term expanded airway organoids from human broncho-alveolar resections to study this disease [86]. These organoids are composed of basal cells, ciliated cells, mucus-secreting cells, and club cells that secret CC10, and they can be utilized in swelling assays to assess CF transmembrane conductance regulator (CFTR) function. Primary ciliary dyskinesia (PCD), another respiratory condition, is caused by dysfunctional airway motile cilia. Building on their earlier work, Clevers et al. optimized airway organoid differentiation into ciliated cells and developed organoids from nasal brushing samples of PCD patients [87]. These organoids exhibited patient-specific variations in ciliary beating patterns that corresponded to the patients’ genetic mutations. COPD is a long-term inflammatory condition affecting the lungs that causes restricted airflow and difficulty breathing. Chotirmall et al. established a method to generate COPD bronchial organoids from tumor tissue, which accurately recapitulated disease-specific features at the individual level (Fig. 3D) [63]. Compared to healthy controls, COPD organoids displayed hallmark features of the disease, including goblet cell hyperplasia and reduced ciliary motility.

Historically, LCOs were primarily established using surgical specimens, which are less feasible for advanced lung cancer cases. Individuals with advanced lung cancer who experience malignant serous effusion (MSE) have a substantially poorer prognosis (the overall survival with and without MSE is 5.49 and 12.65 months, respectively) [88]. Establishing organoids from advanced lung cancer, particularly as models for predicting clinical treatment responses, holds promise for improving therapeutic outcomes in precision medicine. Yang et al. developed a method to successfully generate 214 LCOs from MSE samples of 107 patients [64]. By processing samples into single cells and culturing them with Matrigel, they developed these organoids. Drug sensitivity assays performed on these organoids effectively predicted the clinical treatment outcomes of advanced lung cancer patients, highlighting their potential as a valuable tool for personalized medicine.

Normal lung-derived organoids

When supplied with the right growth factors, adult stem cells (ASCs) extracted from normal tissues have the potential to regenerate and self-assemble into organoids that replicate the structure and function of their in vivo equivalents, comprising various tissue-specific cell types. In 2009, Cleve et al. reported the first generation of organoid-intestinal organoids derived from ASCs [11]. Since then, numerous ASC-derived organoids have been successfully established [89–91]. In 2018, Yuen et al. established ACS-derived human lung organoids and demonstrated their utility by assessing the ability of emerging influenza viruses to infect cells [92].

By randomly combining 3 types of lung cells (bronchial epithelial cells, fibroblasts, and microvascular endothelial cells) and culturing them under 3D support conditions, Tschumperlin et al. generated airway organoids featuring distinct epithelial and endothelial structures (Fig. 4A) [65]. Among them, fibroblasts are vital in preserving the mechanical stability of airway-like organoids formed by cell self-aggregation and may facilitate ongoing interactions with endothelial cells and epithelial cells. Additionally, YAP signaling is essential for the control of epithelial differentiation, as well as the organization and invasion of tubular structures.

Fig. 4 — Protocols for generating of normal lung-derived organoids. (A) The process for establishing airway organoids from adult human lung cells. Reproduced with permission from Ref [65]., Copyright 2017, Elsevier. (B) A protocol for forming hAT2 cells from primary human lung tissues. Reproduced with permission from Ref [93]., Copyright 2020, Cell. (C) A bidirectional differentiation protocol to generate alveolar or airway organoids from primary lung tissues. Reproduced with permission from Ref [67]., Copyright 2023, Bio-protocol

To develop in vitro human AT2 (hAT2) cells model, Lee et al. established a feeder-independent, long-duration 3D culture system to generate alveolar organoids from primary human lung tissue (Fig. 4B) [93]. Imaging and single-cell transcriptomic analyses revealed that the established hAT2 cells model infected with COVID-19 showed fast viral replication and upregulated expression of interferon-related and pro-inflammatory genes. Similarly, Sato et al. established a high-efficiency feeder-independent culture method for human alveolar organoids [66]. These alveolar organoids express ACE2, allowing SARS-CoV-2 to replicate almost 100,000 times within 3 days of viral exposure. Drug testing using these organoids demonstrated that the protease inhibitors lopinavir and nelfinavir, commonly used for treating HIV, exhibited moderate antiviral effects against SARS-CoV-2, whereas the nucleotide prodrug remdesivir showed potent antiviral activity at clinically relevant concentrations.

For bidirectional differentiation of respiratory epithelial cells, Zhou et al. established the first bipotent human respiratory epithelial organoid culture system (Fig. 4C) [67, 94]. This system has the ability to generate of human lung organoids from primary lung tissues that can be passaged, expanded, and cryopreserved. Through induced bidirectional differentiation, the system produces mature alveolar or airway organoids. Experimental findings indicate that the lung organoids can be long-term expansion for more than a year. The differentiated alveolar and airway organoids closely mimic the morphology and function of alveoli and airway, achieving near-physiological levels of human.

Applications of lung organoids

The establishment of lung organoids provides valuable insights into lung development as well as the physiological and pathological processes underlying respiratory health and disease. This information is vital for advancing the prevention and therapy of respiratory-related conditions. In the upcoming sections, we will explore the current and potential applications of lung organoids in modeling early lung development, serving as disease models, and acting as platforms for drug screening.

Early lung development modeling

In early lung development research, mouse models have played a pivotal role [95]. Through these models, researchers have gained insights into critical processes such as the division of the trachea and esophagus and the branching development of the lungs [96, 97]. Furthermore, Gene knockout experiments in mice have further elucidated the signaling pathways involved in lung development [98]. Nevertheless, mouse and human lung tissues differ substantially. For instance, basal cells are distributed across the entire human airway, whereas in mice, they are limited to the proximal airway region [99]. Similarly, goblet cells that secrete mucus are plentiful in human lung tissue but nearly nonexistent in mice [100]. These fundamental differences highlight the limitations of mouse models in fully replicating human lung development. As a result, lung organoids are now a key resource for exploring human lung development. These organoids overcome species-specific differences, enabling researchers to study the mechanisms of lung formation, differentiation, and maturation in a physiologically relevant context.

By combining single-cell techniques with spatial analysis, Rawlins et al. proposed an extensive multi-cell atlas of human lung development (Fig. 5A) [101]. Based on the advanced single-cell and spatial analysis techniques, they discovered 144 distinct lung cell types or states during gestational weeks 5 to 22. By studying the epithelial differentiation proximal-distal gradient, they identified progenitor cells and differentiation states in the developing airways. Their analysis of the mesenchymal revealed 3 distinct niche regions, each defined by unique signaling interactions. These findings provided the foundation for determining the signaling conditions necessary for airway differentiation in embryonic lung. Using this atlas, they functionally validated intercellular interactions critical for alveolar niche development. Notably, Wnt signals from differentiated fibroblasts were shown to promote AT2 cell recognition, while myofibroblasts secreted the Wnt inhibitor NOTUM, establishing a spatial signaling pattern.

Using single-cell RNA sequencing and microscopy, Spence et al. identified a distinct cell type predominantly located in the developing human lung lower airways (Fig. 5B) [102]. These cells co-express SCGB3A2/SFTPB/CFTR and were defined as lower airway progenitor (LAP) cells. Using tissue analysis, organoid models, and single-cell tracing, they demonstrated that LAP cells act as progenitors, producing pulmonary neuroendocrine cells (PNECs) in the airways and a subset of multi-ciliated cells marked by complement component C6. These lineages differ from the basal cell lineage, which generates secretory cells and MUC16-positive multi-ciliated cells. This highlights the diversity of epithelial cells in the lower airways, driven by distinct progenitor populations.

Disease modeling

The global pandemic of COVID-19, triggered by the new SARS-CoV-2 virus, has impacted more than 200 nations and regions, leading to more than 2.5 million deaths. SARS-CoV-2 primarily targets the lungs, and individuals infected with the virus often exhibit respiratory symptoms. Lung organoids have become indispensable in vitro models for investigating the virus’s preference for lung cells and the initial phases of lung infection in recent years [103–105].

Kotton et al. induced AT2 cells from PSCs and used them as a model to research the early apical infection of alveolar epithelium by SARS-CoV-2 (Fig. 5C) [106]. Their findings demonstrated that AT2 cells cultured at the air-liquid interface (ALI) were susceptible to infection by SARS-CoV-2. Transcriptomic analysis of the infected cells revealed rapid changes, including the activation of NF-kB signaling and the disruption of the mature alveolar program, which resulted in a shift toward an inflammatory phenotype. Drug testing using this model validated the efficacy of remdesivir and TMPRSS2 protease inhibitors, reinforcing the suggested mechanism for viral entry into alveolar cells.

Lung organoids are essential for evaluating the infectivity of newly emerging viruses. Using established differentiation methods [86], Kwok-Yung Yuen et al. generated a long-term expandable human airway organoid that includes four distinct types of airway epithelial cells: ciliated cells, goblet cells, club cells, and basal cells [92]. These organoids exhibited synchronous beating cilia, with the proportion of ciliated cells approaching physiological levels. Testing with this model revealed that the human-infecting H7N9/Ah virus replicated more effectively than the less infectious H7N2 virus, while the highly infectious H1N1pdm virus had higher replication titers compared to H1N1sw. These findings demonstrate that airway organoids can differentiate between influenza viruses with varying levels of human infectivity, making them a powerful tool for evaluating newly emerged influenza viruses.

Lung organoids also serve as models for exposure studies, allowing researchers to investigate the biological impact of environmental factors, such as microplastics. Bacchetta et al. quantitatively characterized microplastic fibers (MPFs) released from household dryer exhaust filters and tested their impact on airway organoids (Fig. 5D) [107]. Their results indicated that while MPFs did not restrict organoid growth, they notably decreased gene expression of SCGB1A1, involved in the function of club cell, and altered the directional growth of cells along the fibers. Besides, although MPFs did not induce notable inflammation or oxidative stress, they were enveloped by the cellular layer, embedding fibers within the organoids. This suggests potential long-term implications for alveolar epithelial cell repair.

Drug testing

Standard cell culture systems are cost-effective, high-throughput, and convenient, making them essential for drug screening. Nevertheless, these models frequently fall short of accurately mimicking the intricate physiological and pharmacological responses observed at the organ level. Organoids, to some extent, can capture the characteristics of specific tissues, and have emerged as a promising technology to improve preclinical and personalized drug design. Huang et al. generated a live biobank consisting of 12 LADC organoid lines derived from the most prevalent subtypes of LADC samples, which they applied to anti-cancer drug screening (Fig. 5E) [82]. These LADC organoids retained the genomic traits and overall gene expression patterns of the original parental tumors, making them as reliable models for identifying tumor biomarkers. Based on this model, researchers found that RHOF, SLC16A3, HOXB6, ANXA10, and CDHR1 expression were associated with the survival outcomes of LADC patients, suggesting their potential as prognostic factors. Unlike clinical samples, LADC organoids offer high purity, as they are free from stromal and immune cells, enabling the examination of tumor biomarkers and their influence on tumor behavior without confounding factors. Drug testing on LADC organoids further revealed that compounds targeting the same pathways showed reproducible sensitivity patterns, underscoring their utility in evaluating therapeutic efficacy. The considerable genetic and phenotypic diversity of lung cancer requires individualized treatment strategies. To investigate drug responses, Jang et al. developed a lung organoids model derived from patient tissues, representing five distinct histological subtypes of lung cancer. They found that the drug sensitivity of LCOs correlated with specific genomic alterations: an organoid with a BRCA2 mutation responded to olaparib, an EGFR-mutant organoid was sensitive to erlotinib, and an organoid with both EGFR mutation and MET amplification showed a favorable response to crizotinib [108]. These findings emphasize the promise of lung organoid models in personalizing therapies according to the genetic profile of each patient’s tumor.

Lung-on-a-chip microphysiological system and its application

Single-organ/organoid-on-a-chip

Alveolus-on-a-chip

Alveolus-on-a-chip represents a significant advancement to mimic the architecture and function of the human alveoli for research and therapeutic applications. These micro-engineered systems combine living cells with microfluidic technology to simulate the mechanical, biochemical, and physiological microenvironment of the alveoli.

In 2010, Ingber et al. developed the first lung-on-a-chip system (Fig. 6A) [109, 110]. This biomimetic microsystem successfully replicates the key functional aspects of the human lung, particularly the alveolar-capillary interface. This system consists of two microchannels divided by a flexible, porous membrane. The system replicates the ALI and incorporates mechanical stretching to simulate breathing movements. It has been utilized to study responses to nanoparticles, showing that mechanical strain, such as breathing, can exacerbate toxic and inflammatory responses. Additionally, the chip mimics immune responses in the lung, including the activation of neutrophils in response to inflammatory signals or bacterial infection, simulating the process of immune cell infiltration into the alveolar space. This innovative microsystem presents a new approach for studying lung functions, inflammation, and nanotoxicology, reducing the reliance on animal models while offering more physiologically relevant insights.

In 2015, Guenat et al. described a new lung-on-a-chip system created to replicate the alveolar environment of the lung, including the mechanical strain associated with breathing (Fig. 6B) [111]. This chip features a bio-inspired micro-diaphragm that simulates the contraction of the human diaphragm, inducing cyclic stretching of a flexible, porous membrane. By mimicking the thin alveolar barrier and respiratory movements, the system offers a precise in vitro model of lung mechanics. It supports co-culturing of human pulmonary alveolar epithelial cells and endothelial cells, creating a functional air-blood barrier that closely replicates the physiological interactions. The study demonstrated that cyclic mechanical strain notably affects the epithelial barrier permeability, as well as the metabolic activity and cytokine release (such as IL-8) of lung cells. Thess findings highlight the importance of mechanical forces, such as breathing, in lung function and disease progression. In 2021, the same research group introduced an advanced generation of lung-on-a-chip devices with improved mimicry of the structure and function of human alveoli (Fig. 6C) [112]. Notably, the new design incorporates a biodegradable, stretchable, and biological derived membrane composed of collagen and elastin, offering a more accurate representation of the alveolar membrane’s properties compared to earlier systems that relied on synthetic materials like PDMS. These advancements enhance the physiological relevance of the model, further expanding its potential applications in lung research.

In 2023, Jung et al. presented a novel approach to developing an alveolus-on-a-chip system by integrating high-resolution inkjet bioprinting with microfluidic platforms (Fig. 6D) [113]. This lung model is created using drop-on-demand inkjet bioprinting, which enables precise deposition of lung cells (human alveolar epithelial, endothelial, and fibroblast cells) in a three-layer structure. The bio-printed lung tissues are subsequently cultured on a microfluidic platform, that provides perfusion at the ALI, closely mimicking the natural lung environment. This advanced system allows for enhanced control over flow distribution and enables long-term culture under physiologically relevant conditions. It maintains essential alveolar functions, such as tight junction formation, surfactant protein secretion, and sodium channel activity. Additionally, gene expression analysis shows higher activity of key genes involved in maintaining alveolar barrier integrity and function compared to 2D models. These findings highlight the system’s potential for advancing lung research and providing more physiologically accurate insights into lung function and disease.

Airway-on-a-chip

The airway-on-a-chip technology replicates human lung functions by combining human cells with microfluidic engineering, allowing for the simulation of the lung’s small airways and their responses to various stimuli. This advanced platform has been instrumental in studying lung inflammation, disease mechanisms, and drug efficacy in an environment that mimics physiological conditions, offering an improvement over traditional in vitro approaches or animal model.

In 2015, Ingber et al. introduced a small airway-on-a-chip model, replicating the complex interactions present in the human lung (Fig. 7A) [114]. The chip effectively replicates human lung pathophysiology, especially conditions such as asthma and COPD. When exposed to IL-13, the model exhibits hallmark features of asthma, including goblet cell hyperplasia and reduced ciliary function. Similarly, chips populated with cells from COPD patients demonstrated disease-specific characteristics, including neutrophil recruitment and heightened responses to viral and bacterial infections. This model offers a physiologically relevant system for investigating respiratory diseases and testing therapeutic interventions. By optimizing microfluidic chip design (including stretchable PDMS membranes and collagen IV coating) and culture conditions, Does et al. developed an airway-on-chip platform that simulates the mechanical microenvironment of small airways during normal breathing [115]. Using this chip, they investigated the effects of dynamic fluid shear stress and cyclic stretch on the differentiation and maturation of human primary bronchial epithelial cells (hPBECs), particularly their mucociliary clearance (MCC) function. The results demonstrated that the dynamic chip model significantly accelerated MCC functional maturation compared to traditional static cultures (Transwell inserts), evidenced by enhanced ciliary coordination, a 2-fold increase in mucus transport efficiency, and upregulation of planar cell polarity (PCP) gene VANGL1 expression with altered protein localization. Additionally, dynamic mechanical stimulation reduced inflammatory factors (e.g., IL-8) and extracellular matrix components (e.g., MMP9, fibronectin), suggesting that mechanical forces regulate airway homeostasis. This technology provides a physiologically relevant in vitro model for studying the pathological mechanisms of chronic lung diseases and drug screening, while highlighting the critical role of mechanical cues in modulating airway epithelial function.

Chronic lung diseases, including asthma and COPD, are influenced by intricate interactions between different cell types in the airway tissues. Young et al. created a lung airway-on-a-chip with arrayable suspended gels and investigated the interactions between epithelial cells and smooth muscle cells (SMCs) of human airway (Fig. 7B) [116]. This chip is made up of compartments stacked vertically: a top chamber (for ALI culture of epithelial cells), a middle compartment (contains a suspended hydrogel made of collagen and Matrigel, representing the ECM), and a bottom chamber (for culturing SMCs). Under ALI conditions, epithelial cells form tight junctions and undergo goblet cell differentiation, producing mucus, while SMCs align and express alpha-smooth muscle actin (α-SMA), a marker of contractility. The chip’s thermoplastic design enables mass production and supports the arrangement of multiple systems on a single platform, enabling high-throughput studies. Furthermore, it also enables easy disassembly, which facilitates the extraction of cell cultures for downstream analysis (e.g., protein, RNA). To address the challenge of replicating the epithelial glycocalyx layer, a key player in airway protection and mucociliary clearance, the same group introduced an innovative lung airway-on-a-chip model incorporating bidirectional airflow conditions [117]. This design simulates breathing cycles, successfully promoting the formation and visualization of the glycocalyx layer on airway epithelium for the first time in such a platform. Bidirectional airflow better replicates natural respiratory dynamics, leading to enhanced mucociliary differentiation compared to static or unidirectional flow. Epithelial cells exhibited improved tight junction formation, increased mucus production, and cilia differentiation—features essential for airway function and pathogen defense. This advanced model provides a powerful platform for studying respiratory diseases, testing therapeutic agents, and exploring the role of the glycocalyx in lung health. It offers new opportunities for investigating airway barrier function, mechanisms of viral infection, and the efficacy of treatments targeting lung diseases.

3D cell printing technology enables the automated and efficient fabrication of prototype designs, enabling precise placement of various cell types in specific locations to replicate the natural cellular arrangement of native tissues [118]. Using this technology, Cho et al. created an airway-on-a-chip that closely mimics respiratory tissue (Fig. 7C) [119]. The bioink for the model was derived from decellularized porcine tracheal mucosa, offering a supportive environment for cell proliferation and specialization that closely mimics the natural ECM. This chip integrates airway epithelial cells with a vascular network made up of endothelial cells and fibroblasts, establishing a functional interface between the airway epithelium layer and the underlying vasculature. Such an interface is vital for accurately replicating respiratory functions and immune responses. The model exhibits increased secretion of inflammatory cytokines (e.g., TNF-α) in response to stimuli, highlighting its effectiveness in studying immune responses and respiratory diseases. The system provides a powerful platform for investigating lung physiology, pathophysiology, and potential therapeutic interventions.

While acute inhalation hazard testing has customarily depended on animal models, organ-on-a-chip MPS now offers a viable substitute. Rusyn et al. introduced a human small airway-on-a-chip system to evaluate the effects of inhalation toxicants (Fig. 7D) [120]. The design of the multilayered chip recreates the air-blood barrier of the small airways, allowing for long-term culture (up to 18 days) under physiological conditions while preserving epithelial barrier integrity and ciliary function. The chip was tested with various inhalation toxicants, including lipopolysaccharides (LPS), particulate matter (PM2.5), and iodomethane. Exposure resulted in measurable changes in key indicators, such as increased permeability (barrier function), altered ciliary beating, and elevated levels of biochemical markers like lactate dehydrogenase, effectively replicating respiratory responses observed in vivo. This small airway-on-a-chip offers a robust system for investigating the subacute effects of inhalation toxicants, offering detailed insights into barrier integrity, cellular viability, and immune responses. It represents a significant step toward reducing reliance on animal models while providing physiologically relevant data for respiratory toxicology research.

Lung cancer-on-a-chip

Lung cancer-on-a-chip technology leverages microengineering and cellular biology to create 3D, tissue-like structures that mimic the interactions between lung cancer cells and their surrounding tissue. This approach provides more accurate and physiologically relevant insights into cancer growth, metastasis, and drug responses, providing considerable benefits compared to conventional cells or animal models.

To study metastatic lung cancer, Esfandyarpour et al. introduced a lung cancer-on-a-chip that more accurately represents lung pathogenesis in vitro (Fig. 8A) [121]. This model co-culture of A549 cells and lung fibroblasts within 3D hydrogels formulated to resemble the mechanical and biological aspects of human lung tissue. It features fluidic and air channels to simulate inhalation and exhalation, allowing for a dynamic physiological environment. The model is utilized to investigate disease progression, such as cigarette smoke extract impact on cancer metastasis, and to test the efficacy of anticancer drugs. Developed using 3D extrusion bioprinting, the lung cancer-on-a-chip serves as a powerful preclinical tool for researching lung cancer and the impact of various treatments.

Fig. 8 — Lung cancer-on-a-chip MPS. (A) An in vivo-like 3D lung cancer-on-a-chip MPS. Reproduced with permission from Ref [121]., Copyright 2022, Elsevier. (B) A 3D vascularized lung cancer-on-a-chip MPS. Reproduced with permission from Ref [122]., Copyright 2022, Wiley. (C) A 3D bio-printed vascularized lung cancer organoid-on-a-chip MPS. Reproduced with permission from Ref [123]., Copyright 2023, IOPscience

The tumor microenvironment (TME) significantly affects tumor growth and the effective delivery of drugs through complex interactions. To replicate the TME in vitro, Kim et al. presented a vascularized lung cancer-on-a-chip platform that induces controlled tumor angiogenesis by engineering various TME components, including immune cells, blood vessels, and other elements. (Fig. 8B) [122]. Their study showed that interstitial flow direction influences capillary sprouting, with angiogenesis occurring opposite to the flow. Lung fibroblasts were found to enhance capillary continuity and lumen formation. Thanks to its perfusable vascular networks, this model enhances the transport of anticancer drugs and immune cells to tumor spheroids. By using 3D bioprinting, Jang et al. developed an advanced lung cancer-on-a-chip that combines patient-derived LCOs, lung fibroblasts, and functional blood vessels (Fig. 8C) [123]. A porcine lung-derived decellularized ECM (LudECM) hydrogel was employed in this system to mimic the biochemical makeup of tissues of native human lung. LCOs in LudECM showed greater resistance to targeted anti-cancer drugs that enhance sensitivity compared to those in Matrigel. This vascularized lung cancer system serves a robust platform for assessing drug responsiveness in the context of lung fibrosis, enabling the uncovering of effective treatment options for patients with fibrotic conditions. Additionally, it holds significant potential for developing targeted treatments and uncovering biomarkers for this patient population.

Multi-organ/organoid-on-a-chip

The development of multi-organ/organoid-on-a-chip systems involves creating microfluidic devices that simulate the physiological functions of multiple interconnected human organs. These models aim to replicate the complex interactions between different tissues, allowing for more precise studies of responses of drugs, disease mechanisms, and toxicity assessments. Advancements in this field include the integration of different cell types, including primary human cells, and utilizing biomaterials to replicate the ECM. Researchers are also optimizing flow dynamics and environmental conditions to better reflect in vivo conditions. The ultimate goal is to enhance preclinical testing and reduce reliance on animal models by offering a robust platform for investigating human biology and disease.

The absence of models that closely resemble the integrated and interactive nature of human tissues have resulted in an inability to predict human toxicity of drugs, leading to FDA recalls of approved medications. To tackle this issue, Skardal et al. developed a bioengineered organoids and constructs within a closed-loop circulatory system to support organ communication [124]. They introduced a three-tissue MPS comprising liver, heart, and lung, demonstrating how drug responses can vary based on tissue interactions (Fig. 9A). This emphasizes the significance of incorporating multiple tissues in vitro for studying both the effectiveness and safety profile of candidate medications. Additionally, Skardal et al. developed an integrated multi-organoids-on-a-chip MPS designed to improve drug development efficiency [125]. This platform allows for the simultaneous evaluation of drug effectiveness and safety across multiple tissue-engineered 3D organoids using an affordable, adhesive film-based apparatus. The system requires less than 200 µL of fluid and facilitates matrix-supported 3D cell cultures and spheroid incorporation with a light-activated hyaluronic acid hydrogel. Initially, a three-organoid system featuring liver, cardiac, and lung engineered models demonstrated sustained viability over 21 days. The system was confirmed by examining the metabolism of liver of the prodrug capecitabine into 5-fluorouracil, showing subsequent toxicity in lung and cardiac organoids. The device was later expanded to include six humanized organ models—liver, cardiac, pulmonary, endothelium, brain, and testis constructs. After a two weeks culture period, the interactions between tissues were demonstrated by the liver metabolizing ifosfamide into chloroacetaldehyde, which caused neurotoxicity in the brain organoids. Building on this multi-organoid chip platform, the researchers screened FDA-recalled drugs [126]. The system, featuring various tissue organoids with viability for at least 28 days, effectively detected toxicity for many recalled compounds while maintaining cell viability for non-toxic drugs at clinically relevant doses. Additionally, expanded multi-organoid platforms—including liver, cardiac, lung, vascular, testis, colon, and brain organoids—showed long-term viability and functional biomarker expression (Fig. 9B). The study illustrates how these multi-organoid “body-on-a-chip” devices can model the interconnected metabolism and effects of drugs across different tissues, offering a more physiologically relevant approach to drug screening that could reduce costs and failure rates in drug approval.

Fig. 9 — Multi-organ/organoid-on-a-chip MPS. (A) A three-tissue (liver, cardiac and lung) organ-on-a-chip platform. Reproduced with permission from Ref [124]., Copyright 2017, Springer Nature. (B) A six-tissue (liver, cardiac, testis, vascular, lung and colon) organ-on-a-chip platform. Reproduced with permission from Ref [126]., Copyright 2020, IOPscience

Disease modeling and drug screening

Lung-on-a-chip MPS simulates the physiological environment of human lungs, allowing researchers to explore different facets of lung biology, disease mechanisms, and drug responses. In disease modeling, the systems can be applied to research pathogen interactions (simulating infections by respiratory pathogens, such as viruses and bacteria, to research interactions between host and pathogen and immune defenses), chronic diseases (reconstructing the inflammatory environment and tissue remodeling of chronic lung diseases, such as COPD, asthma, and pulmonary fibrosis, to facilitate studies on pathological mechanisms), and toxicology (assessing the impact of environmental toxins or pollutants on lung tissue, aiding in the understanding of disease progression and risk factors). In drug screening, lung-on-a-chip MPS can be used for efficacy testing (evaluating drug candidates on human lung cells to improve the relevance of results, which offers advantages over traditional animal models), mechanistic studies (helping to identify mechanisms of action for new therapies and predict how drugs affect lung function), and personalized medicine (using cells from patients to advance personalized treatments, tailoring therapies to individual responses).

To investigate within-host viral evolution, Ingber et al. introduced an airway-on-a-chip system to simulate influenza virus evolution, particularly focusing on the emergence of antiviral resistance [127]. This model combines human airway epithelial cells and lung endothelial cells, simulating essential lung functions like mucociliary clearance, immune cell recruitment, and cytokine response (Fig. 10A). By passaging influenza virus across chips upon exposure to antiviral agents (e.g., amantadine and oseltamivir), the study observed the appearance of drug-resistant mutations that are also seen in clinical settings. Specifically, mutations in the M2 and neuraminidase (NA) proteins associated with resistance were successfully modeled. Additionally, the chip was used to replicate gene reassortment events, where genetic material from different virus strains combines, potentially generating new subtypes with pandemic potential. This airway-on-a-chip system also serves as a powerful platform for antiviral drug development and testing [128]. For instance, the study showed that combining nafamostat and oseltamivir extended the treatment window against influenza, and amodiaquine demonstrated prevention and treatment effectiveness against SARS-CoV-2. Moreover, the model accurately reflected clinical outcomes, such as the ineffectiveness of hydroxychloroquine against SARS-CoV-2. By offering a rapid and human-relevant platform, this chip system holds significant potential for advancing research on respiratory viruses, optimizing antiviral strategies, and mitigating the risk of drug-resistant viral strains.

Fig. 10 — Disease modeling and drug discovery. (A) An airway-on-a-chip to study influenza virus evolution. Reproduced with permission from Ref [127]., Copyright 2021, American Society for Microbiology. (B) An airway-on-a-chip to model pulmonary cystic fibrosis. Reproduced with permission from Ref [129]., Copyright 2022, Elsevier. (C) A human lung-on-a-chip to recreate pulmonary edema from drug toxicity. Reproduced with permission from Ref [130]., Copyright 2012, Science. (D) An alveolus-on-a-chip to recapitulate acute radiation-induced lung injury. Reproduced with permission from Ref [131]., Copyright 2023, Springer Nature. (E) An alveolus-on-a-chip to examine the effects of PS-NPs on lung injury. Reproduced with permission from Ref [132]., Copyright 2023, Elsevier. (F) A bionic-lung microfluidic chip to research the response to cigarette smoke exposure. Reproduced with permission from Ref [133]., Copyright 2024, Elsevier

To advance patient-specific treatments and investigate host-pathogen interactions in CF, Ingber et al. created another an airway-on-a-chip device that faithfully replicates key disease features for use in preclinical research [129]. This microfluidic device combines primary human CF airway epithelial cells with lung endothelial cells, simulating the CF lung environment under dynamic fluid flow and an ALI (Fig. 10B). The chip model mimics critical CF characteristics, such as increased mucus production, heightened cilia density and activity, and a pro-inflammatory environment with higher IL-8 levels and increased neutrophil adhesion and transmigration. When infected with pseudomonas aeruginosa, the CF chip showed increased bacterial growth in the mucus, greater inflammatory cytokine production (IL-6, TNF-α, and GM-CSF), and heightened immune cell recruitment compared to non-CF chips. This model offers a robust platform for testing CF therapeutics, providing a human-relevant system to evaluate drug efficacy and better understand the pathological mechanisms of CF.

Pulmonary edema, a condition with fluid congestion in the alveolar regions, impairs breathing and oxygen exchange. To investigate its mechanisms and explore therapeutic options without animal models, Ingber et al. created a lung-on-a-chip device that mimics drug-induced pulmonary edema, specifically interleukin-2 (IL-2)-related edema [130]. This model integrates human alveolar epithelial and endothelial cells that mimics the blood-air barrier and allows the application of cyclic mechanical strain to replicate breathing motions (Fig. 10C). The model replicates leakage of fluid through the blood-air barrier, mimicking the progression of pulmonary edema as observed in patients undergoing IL-2 therapy. Notably, the study found that mechanical ventilation worsens IL-2-induced vascular permeability by widening gaps in cell junctions, identifying a new contributor to pulmonary edema development. The model also facilitated testing of potential treatments, revealing that both angiopoietin-1 (Ang-1) and a TRPV4 ion channel inhibitor (GSK2193874) could prevent IL-2-induced vascular leakage, suggesting new therapeutic avenues. These findings highlight the device as a robust tool for elucidating pulmonary edema mechanisms and identifying new treatment strategies.

Radiation-induced lung injury (RILI) is a condition resulting from high doses of radiation, which can damage lung tissue, particularly following radiation therapy for cancer or exposure to nuclear events. To replicate human lung responses to radiation in vitro and evaluate potential therapeutic drugs, Ingber et al. created an alveolus-on-a-chip system that replicates key aspects of RILI caused by high-dose gamma radiation exposure [131]. This model features human alveolar and pulmonary endothelial cells cultured under conditions that simulate breathing, including cyclic mechanical strain and fluid flow (Fig. 10D). The chip effectively models RILI by reproducing key pathological features, such as DNA damage, enhanced leakage across the alveolar-capillary barrier, cell death, and inflammation, which align with clinical symptoms of pneumonitis and fibrosis. When immune cells (PBMCs) are introduced, the model demonstrates a progressive inflammatory response over several days, marked by elevated pro-inflammatory cytokines. Furthermore, the model enabled testing of drugs like prednisolone and lovastatin. Lovastatin was found to upregulate HMOX1, offering early protection against DNA damage and inflammation; however, prolonged use adversely affected the integrity of the lung barrier. By closely replicating human lung responses to radiation, this system provides a valuable, clinically relevant tool for exploring RILI and screening potential countermeasures, offering significant advantages over traditional animal models.

With increasing environmental pollution, particularly air pollution, research has highlighted the ability of nanoplastics (NPs) and smoke to penetrate deep into the respiratory system, causing oxidative stress, inflammation, and impaired lung function. NPs, specifically tiny plastic particles less than 100 nanometers in size, have been proven to result in lung tissue injury when inhaled. To investigate the impact of NPs on lung tissue in vitro, Liang et al. established a lung-on-a-chip system to examine the effects of polystyrene NPs (PS-NPs) on lung injury [132]. This model integrates immune cells to better simulate human respiratory responses to environmental pollutants at the alveolar-capillary barrier level (Fig. 10E). The study revealed that PS-NPs exposure compromised the functionality of the alveolar-capillary barrier, evidenced by reduced trans-epithelial electrical resistance (TEER) and enhanced permeability. Higher concentrations of PS-NPs were associated with reduced cell viability, increased cell death, heightened oxidative stress, and elevated pro-inflammatory cytokine levels (IL-6, MCP-1, and TNF-α). The model also showed increased immune cell adhesion to endothelial cells, indicating an activated immune response. Importantly, PS-NPs were observed to cross the alveolar-blood barrier, entering the bloodstream and impairing α1-antitrypsin (AAT) expression, a critical marker associated with COPD. This lung chip serves as a powerful tool for environmental toxicology, allowing detailed analysis of pollutant-induced lung injury, oxidative stress, inflammation, and immune responses.

Cigarette smoke (CS), containing thousands of harmful chemicals like tar, nicotine, and carbon monoxide, is another major contributor to lung damage, respiratory dysfunction, and chronic lung diseases. To analyze cellular responses to CS exposure, Xie et al. established a bionic-lung microfluidic chip system [133]. This model integrates BEAS-2B cells and HUVECs cells to assess protective and damaging effects of CS exposure at varying concentrations (Fig. 10F). At lower concentrations of CS, co-culture systems exhibited intercellular protective effects, while higher concentrations led to amplified cellular damage compared to monoculture. The model revealed higher concentrations of reactive oxygen species (ROS) and nitric oxide (NO), biomarkers of oxidative stress and inflammation response. The co-culture model demonstrated dynamic fluctuations in ROS and NO levels, suggesting complex cell-cell interactions. Significant changes in the cGMP-PKG signaling pathway, implicated in inflammatory and oxidative stress responses, were identified as potential modulators of cellular responses to pollutants. Inflammatory and oxidative stress markers, such as IL-8, IL-6, TNF-α, and GM-CSF, were found to peak later in co-culture than in monoculture, underscoring prolonged inflammatory response due to cell interactions. This research demonstrates that the system offers a realistic and adjustable platform for studying cellular responses to inhaled pollutants, providing insights into lung injury mechanisms and potential drug testing for respiratory diseases.

Overall, lung-on-a-chip MPS represents a major breakthrough in preclinical research, providing a more accurate and ethical alternative to traditional models.

Challenges and future perspectives

The development of respiratory MPS has evolved significantly from simple cell cultures to sophisticated models that closely mimic lung physiology. These platforms offer significant potential for enhancing our knowledge of lung biology, disease mechanisms, and therapeutic interventions, demonstrating a promising trend toward replacing human tissues. In the field of OMICS research, their highly biomimetic 3D structures and cellular heterogeneity can provide transcriptomic, proteomic, and metabolomic data that more closely resemble human physiology, substantially enhancing the clinical relevance of genomics, epigenomics, and other multi-OMICS studies [134]. In interdisciplinary applications, the integration of organoids with artificial intelligence enables the construction of “digital twin” models, accelerating advancements in computational medicine. High-resolution live imaging techniques allow dynamic analysis of micro-pathological features in organoids, driving revolutionary progress in imaging diagnostics [135]. In drug development, MPS not only facilitates high-throughput screening of candidate compounds but also enables personalized drug sensitivity testing, offering a new paradigm for precision medicine [136]. This technological framework is propelling life science research from traditional 2D models toward 3D dynamic biomimetic systems.

However, despite their promising applications, respiratory MPS still faces several critical challenges that need to be addressed. The first challenge is the complex physiological structure of the lung. Its intricate structure, including alveoli, airways, and vascular components, is difficult to replicate in MPS. Achieving a realistic 3D arrangement that mimics natural lung tissue is a significant challenge. The second challenge pertains to cell heterogeneity. The lung consists of various cell types, each with unique functions. Accurately modeling this heterogeneity in organoids or chips is essential but difficult, as many systems struggle to incorporate multiple cell types effectively. The third challenge involves functional maturity. Many lung organoids and chips lack the maturity of native lung tissue, which can lead to discrepancies in drug response, pathogen interaction, and physiological functions. The fourth challenge relates to mechanical and physiological stimulation. The lung undergoes mechanical stretching during breathing and experiences fluid flow and gas exchange. Incorporating these dynamic stimuli into microphysiological models remains a challenge. The fifth challenge is reproducibility and standardization. Integration of automated systems for high-throughput screening could facilitate large-scale drug testing and toxicology studies, increasing the utility of respiratory MPS in pharmaceutical research. The sixth challenge is the integration with other organ systems. The lung interacts closely with other organs, particularly in disease states. Developing systems that can mimic these interactions (e.g., lung-heart interactions) is a significant hurdle. Lastly, the seventh challenge is scalability. For widespread application in drug testing or personalized medicine, scalable production methods for organoids and chips must be developed.

Continued research, technological innovations and interdisciplinary collaboration will be vital for overcoming existing challenges. Therefore, in the future, we should concentrate on several key areas to further maximize the potential of respiratory MPS in biomedical research. The first area is advanced materials and technologies. Innovations in biomaterials, such as hydrogels and 3D printing, may enable the development of more complex and functional lung models. Smart materials that respond to physiological stimuli could also enhance realism. The second area is the integration of automated microfluidic technology. Enhanced microfluidic systems can allow for better mimicry of blood flow and air exchange, facilitating more realistic studies of lung function and disease. The third area involves the further development of multi-organ systems. The creation of multi-organ chips that incorporate lung organoids with other organ systems could provide insights into systemic diseases and drug interactions, paving the way for more comprehensive models of human physiology. The fourth area is personalized medicine. Progress in stem cell technology could enable the creation of patient-specific organoids, providing tailored approaches to drug testing and disease modeling. The fifth area is high-throughput screening. Integration of automated systems for high-throughput screening could facilitate large-scale drug testing and toxicology studies, increasing the utility of respiratory MPS in pharmaceutical research. The sixth area is an increased focus on disease modeling. As research progresses, there will be a growing emphasis on using these systems to model specific lung diseases, such as COPD, asthma, and lung cancer, leading to targeted therapeutic strategies. Finally, the seventh area is interdisciplinary collaboration. Increased collaboration among biologists, engineers, and clinicians will be essential for addressing existing challenges and accelerating the development of more sophisticated lung models.

The rapid advancement of MPS has ushered in unprecedented scientific opportunities, while simultaneously raising unique ethical controversies and regulatory challenges. On the ethical front, humanized models (such as human-animal chimeras) may blur species boundaries, while informed consent for patient-derived samples and data privacy require standardization. From a regulatory perspective, current frameworks have yet to clearly classify organoids (as medical devices, biological products, or drug screening tools? ), and issues such as the lack of standardization and the allocation of commercial derivative rights urgently need resolution. Additionally, there are risks of misuse. For instance, gene-edited organoids (e.g., CRISPR-modified tumor models) could potentially be misapplied in bioweapons research, and the integration of organoids with multi-OMICS data may compromise patient genetic privacy. Moving forward, it is imperative to establish an international consensus framework, implement risk-tiered regulations (e.g., restricting high-sensitivity research), and adopt collaborative governance involving multiple stakeholders (scientists, ethicists, patient advocates, policymakers, etc.)—ensuring technological innovation progresses while mitigating biosafety risks and safeguarding healthcare equity.

In summary, while respiratory MPS face significant challenges, their potential for advancing biomedical research and therapeutic applications is vast. Future innovations and interdisciplinary collaboration will be key to overcoming these obstacles and fully realizing the advantages of these technologies.

Acknowledgements

Not applicable.

Abbreviations

MPS: Microphysiological systems
COPD: Chronic obstructive pulmonary disease
OOC: Organ-on-a-chip
ECM: Extracellular matrix
PSCs: Pluripotent stem cells
LPCs: Lung progenitor cells
VAFE: Ventralized anterior foregut endoderm
iPSCs: Induced PSCs
ESCs: Embryonic stem cells
LBOs: Lung bud organoids
AEPCs: Alveolar epithelial progenitor cells
CPM: Carboxypeptidase M
AT1: Alveolar epithelial type I
AT2: Alveolar epithelial type II
HLOs: Human lung organoids
BTOs: Bud tip organoids
LCOs: Lung cancer organoids
NSCLC: Non-small cell lung cancer
PDX: Patient-derived xenograft
LADC: Lung adenocarcinoma
SCLC: Small cell lung cancer
CDX: Cell line-derived xenograft
CF: Cystic fibrosis
CFTR: CF transmembrane conductance regulator
PCD: Primary ciliary dyskinesia
MSE: Malignant serous effusion
ASCs: Adult stem cells
LAP: Lower airway progenitor
PNECs: Pulmonary neuroendocrine cells
ALI: Air-liquid interface
MPFs: Microplastic fibers
hPBECs: Human primary bronchial epithelial cells
MCC: Mucociliary clearance
PCP: Planar cell polarity
SMCs: Smooth muscle cells
α-SMA: Alpha-smooth muscle actin
LPS: Lipopolysaccharides
TME: Tumor microenvironment
LudECM: Lung-derived decellularized ECM
NA: Neuraminidase
IL-2: Interleukin-2
Ang-1: Angiopoietin-1
RILI: Radiation-induced lung injury
NPs: Nanoplastics
PS-NPs: Polystyrene NPs
TEER: Trans-epithelial electrical resistance
AAT: α1-antitrypsin
CS: Cigarette smoke
ROS: Reactive oxygen species
NO: Nitric oxide

Author contributions

Jiaxiang Yin contributed in writing, modifying, and revising. Zirong Bi contributed in writing and reviewing. Tiankai Dai contributed in writing and reviewing. Xiaoyue Zhu contributed in writing and reviewing. Tao Xu contributed in conceptualization and reviewing. Huisheng Liu contributed in conceptualization, reviewing, and revising. All the authors read and approved the final version of the manuscript.

Funding

This work was supported by Major Project of Guangzhou National Laboratory (MP-GZNL2025C03007-01), Young Scientists Program of Guangzhou Laboratory (QNPG23-10), R&D Program of Guangzhou Laboratory (SRPG22-021) and the National Key Research and Development Program of China (2021YFA1101300).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

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Contributor Information

Tao Xu, Email: xu_tao@gzlab.ac.cn.

Huisheng Liu, Email: liu_huisheng@gzlab.ac.cn.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

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PERMALINK

Respiratory microphysiological system in respiratory research: recent advances and future prospects

Jiaxiang Yin

Zirong Bi

Tiankai Dai

Xiaoyue Zhu

Tao Xu

Huisheng Liu

Abstract

Introduction

Table 1.

Lung organoids and its application

Culture techniques

Fig. 1.

Organoids formation

Lung organoids

Table 2.

Pluripotent stem cell-derived lung organoids

Fig. 2.

Patient-derived lung cancer organoids

Fig. 3.

Normal lung-derived organoids

Fig. 4.

Applications of lung organoids

Early lung development modeling

Fig. 5.

Disease modeling

Drug testing

Lung-on-a-chip microphysiological system and its application

Single-organ/organoid-on-a-chip

Alveolus-on-a-chip

Fig. 6.

Airway-on-a-chip

Fig. 7.

Lung cancer-on-a-chip

Fig. 8.

Multi-organ/organoid-on-a-chip

Fig. 9.

Disease modeling and drug screening

Fig. 10.

Challenges and future perspectives

Acknowledgements

Abbreviations

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Data Availability Statement

ACTIONS

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RESOURCES

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