Current and Future Perspectives of Lung Organoid and Lung-on-chip in Biomedical and Pharmaceutical Applications

Abstract

The pulmonary system is a highly complex system that can only be understood by integrating its functional and structural aspects. Hence, in vivo animal models are generally used for pathological studies of pulmonary diseases and the evaluation of inhalation toxicity. However, to reduce the number of animals used in experimentation and with the consideration of animal welfare, alternative methods have been extensively developed. Notably, the Organization for Economic Co-operation and Development (OECD) and the United States Environmental Protection Agency (USEPA) have agreed to prohibit animal testing after 2030. Therefore, the latest advances in biotechnology are revolutionizing the approach to developing in vitro inhalation models. For example, lung organ-on-a-chip (OoC) and organoid models have been intensively studied alongside advancements in three-dimensional (3D) bioprinting and microfluidic systems. These modeling systems can more precisely imitate the complex biological environment compared to traditional in vivo animal experiments. This review paper addresses multiple aspects of the recent in vitro modeling systems of lung OoC and organoids. It includes discussions on the use of endothelial cells, epithelial cells, and fibroblasts composed of lung alveoli generated from pluripotent stem cells or cancer cells. Moreover, it covers lung air-liquid interface (ALI) systems, transwell membrane materials, and in silico models using artificial intelligence (AI) for the establishment and evaluation of in vitro pulmonary systems.

폐는 생리학적 기능과 해부 조직학적 구조 측면을 통합적으로 고려하여 분석해야만 하는 매우 복잡한 조직이기 때문에 폐질환의 병리학적 연구와 흡입독성 평가에 현재까지 주로 동물모델을 사용하고 있다. 그러나 실험동물 윤리와 동물복지를 이유로 점차적으로 실험동물 수를 줄이자는 전세계적인 움직임에 맞춰 생체 외 동물실험 대체법들이 집중적으로 개발되고 있다. 특히 경제협력개발기구(OECD)와 미국 환경보호청(USEPA)은 2030년대 이후, 동물실험을 금지하기로 잠정적으로 합의함에 따라 의생명공학과 제약 분야에서 생체 외 흡입 독성 및 폐질환 모델들을 확립하고 개발된 모델을 이용한 평가 법들의 표준화 연구가 활발하다. 그 모델 중에 예를 들어, 생체칩(organ-on-a-chip, OoC) 및 오가노이드(organoid) 모델은 3차원 바이오 프린터, 미세 유체 시스템, 인공지능(artificial intelligent) 기술들과 접목되어 연구되고 있다. 이러한 생체 장기를 모방한 복합 장기 생체 외 모델링 시스템은 개체 차이를 가지는 생체 내 동물 실험에 비해 복잡한 생물학적 환경을 보다 정확하게 모방할 수 있을 것으로 기대되고 있으나 생체 모방성, 재현성, 민감성, 기반 데이터베이스의 부족 등 아직은 여러 한계점도 가지고 있다. 따라서 본 리뷰 논문에서는 만능성 줄기 세포 또는 암세포를 이용한 폐포, 폐 공기액 인터페이스(air-liquid interface, ALI) 시스템, 트랜스웰 멤브레인(transwell membrane)을 포함하여 폐 OoC 및 오가노이드의 최근 생체 외 폐 시스템 연구결과들과 AI와 접목된 인실리코(in silico) 폐 모델링에 대한 결과들의 현황을 살펴보고자 한다.

Introduction

In 2019, chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma were responsible for 3.97 million deaths, representing a 28.5% increase since 1990 [34]. Furthermore, according to GLOBOCAN 2020 estimates by the International Agency for Research on Cancer, lung cancer causes over 1.8 million deaths annually, making it the leading cause of cancer-related mortality [47]. In addition, recent viral lung infections, like the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, have caused a severe global impact, resulting in 5.7 million deaths within just two years [26]. After severe SARS-CoV-2 infection, the typical pattern of lung disease often includes persistent ground-glass opacity (GGO) and fibrosis bands, which exhibit a mildly restrictive profile, along with abnormal diffusion capacity. Other lung conditions may include organizing pneumonia and severe fibrosis. Due to these respiratory ailments and the SARS-CoV-2 epidemic, the demand for in vitro models has rapidly increased, which is crucial to advancing various therapeutic strategies for lung diseases and understanding complex disease mechanisms.

The human lung is a complex organ responsible for exchanging oxygen and carbon dioxide between the body and the outside environment. It consists of several main components, as depicted in Fig. 1, and its intricate structure serves this vital physiological function. (1) The bronchial tree is the foundation of the respiratory system, originating from the trachea, and branching into primary bronchi that direct airflow to the left and right lungs. Further branching within the lung parenchyma leads to smaller bronchioles, which eventually form microscopic air sacs called alveoli. (2) Alveoli, resembling clusters of tiny grapes, are crucial sites for gas exchange, where oxygen diffuses across thin alveolar walls primarily composed of type I and type II cells, while carbon dioxide moves in the opposite direction for exhalation. (3) Bronchioles, characterized by cartilaginous structures and smooth muscle linings, play a crucial role in regulating airflow dynamics within the lungs, directing air to various lung regions. (4) Alveolar ducts and sacs, located at the terminations of bronchioles, act as conduits for air delivery to the alveoli, with clusters of alveoli arranged in alveolar saccules at the duct ends. Despite the remarkable complexity of its structure and function, in vitro cell culture systems face challenges in replicating the differentiation polarity observed in vivo respiratory system, particularly among cells in direct contact with air. Immersion in a cell culture medium fails to mimic the physiological environment, hindering accurate imitation of developing structures and limiting the ability to evaluate inhalation toxicity.

Fig. 1. Human bronchial tree. Lobule, and alveolar sac anatomy.

In recent years, there has been notable progress in the development of highly differentiated respiratory and lung models, offering valuable insights into humoral and cellular responses at barrier sites. However, despite their advancements, these models often lack essential components such as immune cells, humoral immune factors, and lung-associated microflora. Moreover, the diversity of human diseases and their corresponding disease in vitro models presents a significant challenge.

In this manuscript, our objective is to investigate the current landscape of respiratory and pulmonary in vitro models, with a specific focus on organoids, Organ-on-a-chip (OoC) systems, ALI cultures, 3D bioprinting, and in silico modeling. We comprehensively reviewed their cultural implications, progress, and anticipated challenges in advancing these cutting-edge technologies in the future.

Advancements in alternatives to animal testing for inhalation toxicity assessment: a historical perspective

To date, inhalation toxicity tests have mainly utilized experimental animals. However, it is difficult to transfer research findings to human pathophysiology because laboratory animals are also unable to fully reproduce human lung anatomy, cellular differences, and environmental exposures [2]. As the application of the 3R principles expands and understanding of animal ethics grows, U.S. lawmakers and human welfare organizations are increasing pressure to reduce animal testing, while encouraging the development of alternative in vitro animal testing methods [23]. However, although the development of alternative animal testing methods for inhalation toxicity testing, which began in the 1980s, has been steadily progressing, there is still no standardized inhalation toxicity testing model.

Emergence of the air-liquid interface (ALI) model

The Air-Liquid Interface (ALI) system represents a valuable tool designed to emulate physiological environments like the respiratory tract and skin, facilitating cell exposure to both air and liquid phases. The inception of the ALI system traces back to 1975 when Michalopoulos et al. [30] established efforts to culture adult rats primarily cultured liver tissue on a floating collagen membrane to enhance cell survival [30]. They hypothesized that proximity to the air layer promoted efficient oxygen exchange, thus prolonging cell viability [30]. After this pioneering work, several research teams endeavored to culture cells at the interface of the culture medium and air layer (Fig. 2) [12, 13, 37]. Lung-on-a-chip (LoC) platforms enable the replication of physiological flow dynamics, encompassing both blood circulation in the basal compartment and air movement. Additionally, these platforms can simulate the submucosal layer. Furthermore, lung-on-a-chip models facilitate the administration of aerosols to simulate the inhalation of droplets or particulate matter [4].

Fig. 2. Air-liquid interface configurations. (A) Standard 2D ALIs and (B) ALI in lung-on-a-chip devices operated in semi-dynamic or dynamic mode [4].

The first study reported that an attempt to culture respiratory epithelial cells using the ALI approach was undertaken by Whitcutt et al. in 1988 [52]. Their experimentation revealed notable outcomes, including fluid secretion and differentiation into a ciliary columnar morphology when cells were cultured under ALI conditions. This phenomenon promoted a mucociliary phenotype reminiscent of the in vivo mucociliary layer. Building upon this foundation, numerous studies have reaffirmed that airway epithelial cells cultured at the ALI retain their ability to differentiate into mucus-secreting and ciliated cells, closely resembling the in vivo phenotype of tracheal epithelium [14, 19, 21]. Additionally, the ALI system offers distinct advantages, particularly in detecting inhalation toxicity responses effectively. By preserving the physicochemical properties of airborne particles and preventing particle-medium interactions [5, 25, 42], such as partial dissolution, inherent to submerged conditions, the ALI system emerges as a robust platform for toxicity assessment.

In the 1990s, ALI systems were intensively studied, driving active research toward the development of efficient exposure systems. In 1993, Chen et al. [7] pioneered ALI simulation using fibronectin-rich permeable membranes and inertial collisions to deliver sulfuric acid aerosols of uniform size and mass concentration to target cell monolayers. This innovative approach provided valuable insights into the mechanisms underlying aerosol-induced respiratory effects [7]. In 1995, Tu et al. [49] devised a sulfuric acid aerosol system capable of rapidly adjusting concentration levels within 5 min and maintaining consistency for up to 60 min, while also ensuring a humidified environment to prevent cell desiccation. Their reverse monolayer exposure system aimed to alleviate cell drying issues [49]. Additionally, Muckter et al. [32] introduced the first polycarbonate-based device capable of adjusting flow rates and precisely controlling exposure environments by monitoring chemical vapor concentrations [32].

Furthermore, exposure systems of in vitro respiratory platforms aimed to closely replicate in vivo conditions. In 2000, Aufderheide et al. [1] introduced the CULTEX system, which mimics the wetting and drying cycle of the respiratory epithelium through intermittent medium supply [1]. In 2002, Tippe et al. [48] proposed an aerodynamic exposure system transporting aerosol particles near the cell surface, allowing settling by convection, Brownian diffusion, and gravity [48]. The system addressed the challenge of particle dosimetry by developing a novel method to calculate aerosol particle settling in the cell culture medium [48]. In 2003, Detlef et al. [39] devised a method to mimic actual respiration using intermittent exposure conditions [39]. In 2008, Diabaté et al. [10] validated an in vitro test system for assessing the toxicity of complex environmental atmospheres encountered by human lung cells, employing intermittent exposure conditions, particularly diluted cigarette smoke, to emulate real breathing [10]. Furthermore, co-cultured human lung cells in transwell inserts were exposed to aerosols to mimic lung particle deposition [10]. In 2010, it was demonstrated that to faithfully replicate the in vivo environment, the ALI system should employ human airway epithelial primary cultured cells instead of cell lines [36]. Khoufache et al. [22] developed an in vitro model using porcine primary cultured tracheal epithelial cells, less legally restricted and more readily available for ALI. This model accurately replicated airway epithelium responses to Aspergillus fumigatus conidia or fungal toxins, validated against in vitro monolayer human cell culture studies [22].

Organ-on-a-chip (OoC)

Organ-on-a-chip (OoC) represents an in vitro model constructed on a microfluidic chip, aiming to faithfully replicate the structural, functional, and mechanical aspects of specific organs or organ systems within the chip. Table 1 provides an overview of the cells, extracellular matrix (ECM), and materials utilized in OoC systems (Fig. 3). The conducting airways feature a pseudostratified epithelium, consisting of various cell types such as mucus-producing goblet cells, ciliated cells, neuroendocrine cells, and basal cells. In contrast, the alveolar epithelium is composed of flattened type 1 epithelial cells and cuboidal type 2 epithelial cells. Within the submucosal layer of the airways reside interstitial and vascular cells, while the alveolar-capillary interface showcases the proximity of the epithelium to the endothelium [4].

Fig. 3. Cells of the lung. Various cell types composed of the each different airway; trachea to bronchi, bronchioles to alveoli, and alveoli [4].

Table 1. Specifications of OoC system

In 2010, the innovative work of Huh et al. [17] introduced the first LoC, designed to mimic the human alveolar-capillary interface (Fig. 4). In their model, human alveolar epithelial cells were cultured on one side of a membrane coated with ECM, while pulmonary microvascular endothelial cells were cultured on the opposite side [17]. A distinctive feature of this model is the application of vacuum to the microchamber, causing elastic deformation of the flexible polydimethylsiloxane (PDMS) to simulate real respiratory movements. Subsequent studies utilized this platform to develop a model of pulmonary edema disease treated with IL-2, demonstrating the therapeutic efficacy of GSK2193874, a novel drug previously validated in animal experiments, thus highlighting the potential of LoC as an alternative to traditional animal testing methods [16]. Two years later, Long et al. [27] devised an optimized liquid phase flow pattern for LoC through computational microfluidic simulations and quantitative measurements of dye concentrations at the chamber inlet and outlet [27]. These efforts minimized stagnation zones, ensuring consistent exposure of cells to nutrients and drugs while enhancing the predictive accuracy of in vitro models.

Fig. 4. Interfaces and dimensions of LoC devices [4].

A microfluidic device known as a LoC (depicted in Fig. 4) offers the versatility to accommodate monoculture, co-culture, or tri-culture arrangements of cells, distinguished by their 2D, 2.5D, or 3D configurations on supporting scaffolds [8]. Monocultures, although not depicted, exclusively comprise the epithelial layer, as seen in (A). Within (A), epithelial cells form a monolayer on the upper side of a permeable membrane, juxtaposed with endothelial cells forming a monolayer on the lower side. Similarly, in (B), epithelial cells occupy the upper side of a permeable membrane, while fibroblast cells occupy the lower side. Both scenarios in (A) and (B) exhibit vertically aligned monolayers, coinciding with the optical axis of the objective lens during microscopy. In contrast, (C) demonstrates a different arrangement, with epithelial and endothelial cell monolayers cultured on a hydrogel, oriented perpendicular to the optical axis of the objective lens. (D) presents a tri-culture setup, where stromal cells and endothelial cells are segregated by two permeable membranes. In (E), the tri-culture configuration includes an epithelial cell monolayer situated above a 3D gel containing embedded stromal cells and vascular endothelium. Finally, (F) depicts lumens lined with cells within a 3D gel embedding stromal cells. Here, epithelial cells form a single layer lining an air-filled lumen, while endothelial cells line a blood/media-filled lumen [4].

Later, a model introduced by Sellgren et al. [41] replicated the architecture of the airway mucosa [41]. Their model comprised three vertically aligned compartments separated by nanoporous membranes, with airway epithelial cells cultured in the ALI compartment and fibroblasts and lung microvascular endothelial cells cultured in the adjacent compartments. Notably, a novel coupling technique was developed to apply hydrophilic polytetrafluoroethylene (PTFE) membranes, creating an optimal environment for airway epithelial cell growth. This advancement enhanced physiological mimicry and enabled autonomous operation for over 3 days without an external power source. In addition, a study by Stucki et al. [45] presented a LoC model that emulates the lung parenchymal environment by incorporating a micro-diaphragm to mimic in vivo diaphragmatic movements [45]. This model consists of two PDMS plates separated by a thin, porous, flexible PDMS membrane. A 40 µm PDMS layer with pneumatic channels is bonded to one of the plates, mechanically stretching the PDMS membrane to simulate breathing motions. Utilizing this device, bronchial epithelial cells were cultured on thin, porous PDMS membranes and subjected to periodic deformation to assess their impact on cell permeability. The study confirmed an increase in small molecule transport without compromising barrier integrity. Additionally, cyclic elongation was found to influence the metabolic activity and cytokine secretion of primarily cultured human alveolar epithelial cells.

In the year preceding the SARS-CoV-2 epidemic, Zhang et al. [58] introduced an innovative 3D human LoC model tailored to replicate the structure and function of the human alveolar-capillary barrier for evaluating nanoparticle pulmonary toxicity [58]. This model consists of three parallel channels where human umbilical vein endothelial cells (HUVECs) and human alveolar epithelial cells are co-cultured surrounding a Matrigel membrane. The upregulation of E-cadherin and VE-cadherin expression, pivotal for the selective permeability of the barrier, suggests the emulation of critical features of the alveolar-capillary barrier. Notably, the incorporation of cell-cell, cell-substrate, and vascular mechanical signaling synergistically enhanced barrier function, thereby more authentically mimicking the human lung.

Conversely, Humayun et al. [18] focused on elucidating the interactions among airway smooth muscle cells, epithelial cells, and the extracellular matrix (ECM) [18]. They introduced a novel thermoplastic-based lung airway-on-a-chip model designed with three vertically stacked microfluidic compartments made of poly (methyl methacrylate) (PMMA). These compartments include a lower reservoir for the culture of smooth muscle cells (SMCs), a thin hydrogel layer serving as an intermediate biocompatible matrix, and an upper chamber for ALI culture of epithelial cells. This design facilitates the investigation of interactions between SMCs, epithelial cells, and the ECM. PMMA, chosen as the body material, offers bio-inert, compatibility with mass production, low cost, optical transparency, leak-free binding, and preservation of microscale characteristics. Moreover, Yang et al. [55] utilized poly (lactic-co-glycolic acid) (PLGA) electrospun nanofiber membranes to develop LoCs [55]. PLGA nanofiber membranes are deemed biocompatible, enabling cells to grow and interact similarly to the in vivo respiratory environment. These membranes provide a porous and permeable scaffold suitable for mimicking the alveolar respiratory membrane and can closely emulate the in vivo environment by adjusting their thickness.

In 2020, Schimek et al. [40] pioneered a human multi-organ co-culture system that integrates a 3D bronchopulmonary model with liver spheroids to delve into the systemic impacts of substance exposure, with a keen focus on the interplay between lung and liver tissues [40]. This system was meticulously crafted to dissect the ramifications of substance exposure on the entire organism. Leveraging chip3plus, an enhanced version of the HUMMIC Chip platform tailored for multi-organ co-culture investigations, the system comprises three distinct culture compartments arranged sequentially. The initial compartment adopts a 96-well plate format, widely employed for high-throughput screening, while the remaining two compartments are configured in a 24-well plate format, facilitating larger and more comprehensive tissue culture. Spheroids were strategically positioned in these compartments to foster interaction between lung and liver tissues. Upon subjecting the optimized system to the hepatotoxic and carcinogenic agent aflatoxin B1 (AFB1), the protective effect of liver spheroids on lung tissue integrity became apparent, contrasting with the dysfunction observed in monolayer-cultured bronchial mucosal/airway tissue. Furthermore, exposure to AFB1 prompted a notable reduction in transcutaneous electrical resistance and an increase in lactate dehydrogenase (LDH) release in monolayer-cultured bronchial mucosal/airway tissue. In sharp contrast, lung-liver co-cultures exhibited stable intracellular ATP levels and no significant increase in LDH release, indicative of preserved liver function [40].

Noteworthily, in 2021, Zamprogno et al. [56] developed a membrane used for LoC, with physiologically relevant components [56]. Using the principle of surface tension, they fabricated collagen-elastin (CE) membranes with rat-tail type 1 collagen and bovine neck elastin. A drop of CE solution was injected into a 2 mm diameter, 18 µm thick gold mesh using a micropipette, and after gelation using surface tension, the mesh was dried at room temperature for about 2 days. The thickness is about 10 µm, and once made, it can be stored for about 3 weeks. The cells are soaked in the media 2 hr before seeding before use. A comparison of the performance of the CE membrane and PET membrane through spectrometry revealed that the CE membrane absorbs only 10% of visible light, while the PET membrane absorbs 20%. In addition, when the membranes were treated with a certain concentration of matrix metalloproteinase 8 (MMP8) to see the biodegradability of the membranes, it was confirmed that the membranes were completely degraded without any residue, although the degradation time was inversely proportional to the concentration. This means that CE membrane performs better in the optical property department and has better biodegradability. 10 µM Rhodamine B was compared to PDMS and PET membranes and small molecule absorption and adsorption. The absorption and adsorption of small molecules were about 90% lower in the CE membrane than in other membranes, and the problem of absorption/adsorption of small molecules, which is a problem of PDMS, was alleviated by about 90%. In addition, membrane permeability, stretchability, and tunability were also tested. Human primary alveolar epithelial cells (hAEpCs) and human lung microvascular endothelial cells (VeraVec) were successfully cultured on CE membranes, and when Lung alveolar epithelial cells were cultured together, a functional barrier was also observed to be formed successfully membranes, unlike PDMS membranes, no separate pre-coating was required, and hAEpCs and VeraVec could be cultured for at least 3 weeks [56].

Recent years have witnessed remarkable progress in OoC-related technologies, culminating in the creation of organoids that faithfully mimic a wide array of biological organs. Furthermore, the integration of LoC with these organoids in multi-organoid configurations has been achieved (Fig. 5) [54]. The introduction of the concept of ALI has further propelled this field forward, allowing for the fusion of diverse membrane materials and microfluidic systems to replicate the intricate dynamics of air and capillary flow, reminiscent of living lung tissue. These revolutionary in vitro models exhibit exceptional sensitivity, offering invaluable tools for advancing the development of novel therapeutics for pulmonary diseases and conducting comprehensive evaluations of inhalation toxicity. Importantly, these systems are available in a variety of formats, catering to the diverse research needs of investigators.

Fig. 5. Design of the biologically inspired biomimetic multi-organ microfluidic chip system [54].

Fig. 5 presents a multi-organ chip system, detailing various components: (A) depicts an illustration of lung cancer metastasis to multiple distant organs [8]. (B) offers a schematic representation of the multi-organ microfluidic chip, featuring an upstream "lung organ" and three downstream "distant organs." (C) outlines the assembly process, where three polydimethylsiloxane (PDMS) layers are aligned and bonded irreversibly to create two sets of three parallel microchannels, separated by a PDMS membrane containing an array of through-holes with a diameter of 10 μm. (D) describes the culture of human bronchial epithelial and stromal cells in two parallel microchannels divided by a membrane. In (E), the bronchial epithelial cell layer interacts with air in the upper channel, while the culture medium flows through the microvascular channel. (F, G) illustrate the recreation of physiological expiration movements through the application of a vacuum to the side chambers, inducing mechanical stretching of the PDMS membrane. (H) highlights the co-culture of lung cancer cells with human bronchial epithelial cells. (I) shows the 3D culture of cells from multiple organs in dedicated chambers. Lastly, (J) depicts the flow of the culture medium through the microvascular channel, simulating in vivo blood circulation [54].

Integration system of organoids and OoC

Advancements in the in vitro respiratory platform models have focused on OoC and organoid fusion systems. In 2017, Skardal et al. [44] engineered a multifunctional OoC system comprising liver, heart, and lung components [44]. The lung modules were developed using three different cell types cultured on transwell membranes, while liver and heart organoids were cultured separately and subsequently integrated into the chip. Microfluidic devices were constructed using PDMS material, with lung fibroblasts, epithelial cells, and endothelial cells cultured in porous PET membranes to form threelayered lung modules.

Expanding upon this methodology, in 2020, Skardal et al. [43] developed a chip consisting of glass, double-sided tape, polymethyl methacrylate (PMMA), and polycarbonate layers, incorporating liver, heart, lung, blood vessels, and testis organoids. Brain organoids were also loaded onto the chip and fused with other organoids to create an integrated OoC platform. This innovative approach enabled toxicity testing of recovered drugs and their metabolites [43]. Moreover, Van et al. [50] employed magnetic-activated cell sorting (MACS) to selectively culture cells expressing HTII-280, a marker indicative of alveolar type 2 (AEC2) cells. These cells play critical roles in ion transport, epithelium repair, and immune defense against microorganisms within the lung tissue. Expression and proliferation of markers associated with AEC2 (HTII-280, SP-C, E-cadherin) and AEC1 (RAGE, Aquaporin, vimentin) characteristics were confirmed across various culture conditions, including transwell inserts, organoids, and OoCs. The successful isolation of AEC2 cells demonstrated their sustained expression of characteristic markers over time [50]. Furthermore, while lung organoids derived from patient cells have demonstrated retention of genetic characteristics and suitability for in vitro personalized drug screening, they still face limitations in representing the diverse growth patterns and heterogeneity observed in lung cancer [8, 28]. Notably, these patient-derived organoids predominantly consist of epithelial cells, lacking the inclusion of immune or stromal components necessary for modeling the complex microenvironment of the lung.

Conversely, OoC platforms have emerged as powerful tools enabling visualization and quantitative analysis of various biological processes within intact lung organs, a feat unattainable with traditional cell cultures or animal models [16, 17]. These platforms facilitate the reconstruction of numerous physiological functions and allow for the study of intricate physiological phenomena. For instance, Xu et al. [53] microfluidic chip-based 3D coculture model demonstrated the feasibility of individualized drug therapy for lung cancer patients, enabling the use of drug dose and efficacy to aid in chemotherapy selection [53]. Additionally, Zhang et al. [57] successfully established a human disease model of SARS-CoV-2 infection on a chip, reproducing immune responses observed clinically and shedding light on the pathogenesis of severe COVID-19-associated microvascular thrombosis [57]. This underscores the potential of OoCs to replace animal models for evaluating and repurposing drug candidates for SARS-CoV-2 treatment. Thus, the integration of lung organoids with LoC technology holds promise for addressing the limitations of organoids, particularly in terms of reproducibility and fidelity in mimicking lung tissue.

Integration system of OoC and bioprinting technology

Das et al. [9] developed a lung cancer OoC model (IVM3DLCOC) that can be mimicked in vivo system by utilizing 3D bioprinting [9]. It consists of a lid, air channel, porous layer, and fluid channel for a total of four layers, and PDMS was used as the material for both the chip and membrane. When A549 (human lung cancer cells) and human lung fibroblasts were bio-printed on the OoC and treated with cigarette smoke extract (CSE), the expression of N-Cad, a metastasis marker of A549, increased about 3-fold, and the expression of α-SMA in human lung fibroblasts. The expression of N-Cad, a metastasis marker of A549, was increased about 3-fold, and that of α-SMA was increased about 2.5-fold in human lung fibroblasts. When CSE was treated with IVM3DLCOC, there was an observed increase in the expression of interleukin-6 (IL-6), a marker of pulmonary fibrosis, as well as a promotion of epithelial-mesenchymal transition (EMT) associated with the metastatic potential of lung cancer cells. These results validate the function of IVM3 DLCOC in human lung cells under disease and normal conditions and demonstrate the applicability of this model for mechanistic analysis of disease and evaluation of anticancer drug effects [9].

In contrast to organoids, which typically adopt a simple spheroid structure composed of cells, and conventional monolayer cell cultures, which exhibit some limitations in recapitulating in vivo cellular functions, advanced OoC platforms offer a remarkable opportunity to emulate physiological conditions. A study conducted by Stucki et al. [45] showed that primary human alveolar epithelial cells cultured in a dynamic mode of respiration exhibit heightened metabolic activity, compared to cells cultured in a static environment [45]. Additionally, investigations by Moura et al. [31] demonstrated that microfluidic approaches within OoC systems enable precise channeling of nutrients and oxygen to cellular systems. This microfluidic network effectively removes metabolic waste products that may accumulate in static cultures. The implementation of perfusion flow in OoC platforms not only mimics the natural vascular environment more accurately but also provides shear stress stimulation to cells in the lung. Moreover, OoC systems require fewer cells and reagents, resulting in cost reductions and contributing to overall affordability [31]. This integrated approach of OoC platforms could provide a promising tool for advancing in vitro respiratory-related research methodologies.

In 2023, a study by Kim et al. [24] employed inkjet bioprinting to develop a multiplexed chip based on PDMS material featuring four holes, microfluidic channels, and culture inserts mimicking lung structure and function [24]. The membrane utilized in this chip was constructed from polycarbonate. Evaluation of lung tissue cultured on the chip revealed functional characteristics and expression patterns of biomarkers associated with alveolar barrier function (Fig. 6). Structural analysis indicated that the chips were approximately 8 µm thick, with cell viability comparable to that of conventional plate cultures as determined by the CCK-8 assay. The authors assert that this model represents the thinnest in vitro alveolar model reported to date, capable of supporting robust cell viability. Immunofluorescence analysis and qPCR confirmed the expression of proteins and genes associated with alveolar barrier tissue function. Notably, tight junction protein zonula occludens-1 (ZO-1) and surfactant protein A (SP-A) were observed in the intercellular region, indicative of typical functional properties of the alveolar barrier. Additionally, expression levels of epithelial sodium channels (α-ENaC, β-ENaC, and γ-ENaC), Na+/K+ transport ATPase subunit α 1 (ATP1A1), and surfactant genes (SP-A and SP-B) were significantly elevated in the biochip model compared to 2D monolayer cell culture conditions. These findings underscore the adaptation of bioprinted alveolar barrier tissue by epithelial cells, efficient surfactant secretion, and adequate expression of genes essential for alveolar epithelium function.

Fig. 6. Schematic images of the microfluidic system for mounting culture inserts containing inkjet-bioprinted alveolar barrier tissues [24].

The microfluidic system depicted in Fig. 6 can be described as follows: (i) Utilization of a PDMS culture insertmountable biochip, (ii) application of inkjet bioprinting to introduce human alveolar living cells into a tissue culture insert provided commercially, (iii) incorporation of a living tissue-containing culture insert into a PDMS chip through mounting, and (iv) perfusion of culture medium to the lung-on-a-chip setup post-mounting of four inserts. In addition, (B) includes (i) A schematic cross-section image illustrating a lung-on-a-chip, incorporating a fluidic system, and (ii) a schematic cross-section image depicting the alveolar barrier tissue on a chip [24]. Furthermore, Grigoryan et al. [15] achieved the reproduction of 3D transport through the vascular network, a challenging task, utilizing 3D bioprinting technology. This advancement enabled the study of oxygenation and red blood cell flow. Notably, 3D bioprinting facilitated the simultaneous deposition of cells and materials, enabling precise cell placement within the biomaterials [15].

Integration system of OoC and AI technology

Recently, in silico technology-based experiments using AI are also underway. Among them were the results of Kanda et al. [20] which optimized the cell culture process more rapidly and increased the quality and number of cell products through a system that combines robotics and artificial intelligence [20]. The results are expected to be useful in the process of culturing induced pluripotent stem cells, inducing differentiation to make organoids, and placing cells in OoCs. Bian et al. [6] also performed the first experiment applying deep learning to detect and track organoids [6]. After detecting organoids through a deep neural network (DNN), they used the DNN to track the organoids and saw if toxicity and drug response tests affect the organoid’s genetic and proteomic status quickly and accurately (Fig. 7).

Fig. 7. A schematic overview of integrating OoC and AI systems.

In the research conducted by Mencattini and colleagues, the image analysis phase is carried out autonomously by AI, ensuring an unbiased process. Therefore, the effects of various treatment combinations and schemes (dosage, injection sequence, etc.) on anti-tumor cytotoxicity and immune cell behavior can be accurately measured in LoC [29]. Although there were no data yet on experiments directly applying AI, a study by Paek et al. [35] on bone-on-a-chip confirmed that bone-on-a-chip is superior to the transwell method in terms of improved cell function, and that fluorescence AI-based image analysis with fluorescent staining after drug testing confirmed its high accuracy and stability [35]. This means that even LoC can perform toxicity testing based on high accuracy and stability if fluorescent staining is performed after drug testing and image analysis is performed, which is expected to be a great help in immunotherapy and personalized tailor-made drug development shortly.

Conclusion

The lung is an intricate organ consisting of branches that connect the major airway with millions of small gas exchange units. Traditional pulmonary biomedical research and inhalation toxicity tests relying on cell line models face limitations due to a lack of cellular diversity. In vivo animal models also present challenges including ethical concerns and variations between races and physiological conditions. The reduction of animal experimentation is currently in high demand. To address these issues, organoids and OoC models provide promising solutions. Organoids are 3D self-organized structures made up of various lung cells derived from stem cells and cancer cells cultured with essential growth factors. OoC models are biomimetic microsystems customizable to simulate blood flow or human breathing using microfluidic systems. Furthermore, the integration of OoC and ALI platforms, AI algorithms, in silico modeling, and advanced machine learning techniques is a critical advance in respiratory disease research and respiratory toxicity assessment. This multidisciplinary approach provides an unprecedented opportunity to simulate complex biological systems at high fidelity and gain greater insight into the nature and behavior of cellular properties, disease mechanisms, and drug response. It will also accelerate the development of treatments for lung diseases and improve predictive capabilities, ultimately contributing to the development of safer and more effective therapies. With the improvement and integration of these innovative technologies, the future of respiratory disease research and respiratory toxicity assessment has great potential to revolutionize personalized medicine and improve human health. Given their significance and advantages, we anticipate that these model systems are anticipated to offer a superior platform for biomedical researchers studying pulmonary diseases and inhalation toxicity tests, including emerging viral infections (e.g., SARS-CoV-2, coronavirus disease-19) progressive fibrotic pulmonary diseases, and primary or metastatic lung cancer.