Three-dimensional lung models are laboratory-built systems that recreate the architecture, cell diversity, and mechanical behavior of living lung tissue in ways that flat cell cultures and animal experiments cannot. They range from tiny chips lined with breathing human cells to miniature organoids grown from a patient’s own stem cells, and they are reshaping how researchers study respiratory disease, test drugs, and evaluate inhaled toxins. The field has advanced rapidly since the first “lung-on-a-chip” was described in 2010, and recent regulatory changes in the United States have given these models an even larger role in pharmaceutical development.
Why Flat Cell Cultures Fall Short
For decades, the workhorse of lung research was a layer of cells growing on the bottom of a plastic dish. These two-dimensional cultures are cheap and fast, but they strip away most of what makes lung tissue behave like lung tissue. Cells in a flat dish adopt an unnatural shape, lose their normal tissue architecture, and express different proteins than cells in a living lung.1Nature. Lung adenocarcinoma cells respond differently to mechanical stress in 3D versus 2D environments They never experience the rhythmic stretching of a breath or the flow of blood past their surface. The result is data that sometimes misleads: a drug that kills cancer cells on a dish may do nothing inside a tumor, and a toxin that seems harmless in 2D may provoke a fierce inflammatory response in a more realistic setup.
Animal models fill in some of those gaps, but mouse lungs differ from human lungs in cell composition, immune signaling, and airway geometry. These differences have contributed to a long history of drugs that look promising in rodents but fail in human clinical trials. Three-dimensional lung models were developed specifically to close this gap, giving researchers systems that are human, structurally realistic, and controllable enough to run repeatable experiments.
Air-Liquid Interface Cultures
The simplest form of 3D lung model is the air-liquid interface, or ALI, culture. Instead of submerging cells in liquid, researchers grow airway epithelial cells on a porous membrane. The top surface of the cells is exposed to air while the bottom receives nutrients from a liquid medium underneath. This arrangement mirrors the real lung, where the inner lining faces inhaled air on one side and blood vessels on the other.2PubMed Central. Protocol for differentiating primary human small airway epithelial cells at the air-liquid interface
Given a few weeks in this setup, the cells differentiate into the specialized types found in a real airway. They form a tight, electrically resistive barrier, secrete mucus, and even grow functional cilia that sweep particles along the surface, just as they would inside your nose or bronchi.3PubMed Central. Air-Liquid Interface Cultures to Model Drug Delivery through the Mucociliary Epithelial Barrier More advanced versions embed native lung fibroblasts and solubilized lung matrix proteins beneath the epithelial layer, which further pushes the cultures to mature into goblet cells, club cells, and ciliated cells with proper barrier function.4Scientific Reports. Development of a novel air–liquid interface airway tissue equivalent model for in vitro respiratory modeling studies
ALI cultures have become a standard platform for inhaled drug testing and toxicology because you can deposit aerosols directly onto the cell surface, much as particles land in a real airway. The trade-off is that they are relatively static: no blood flow, no immune cell circulation, and no breathing motion. For those features, researchers turn to more complex systems.
Lung-on-a-Chip Systems
A lung-on-a-chip is a microfluidic device, typically about the size of a USB stick, that recreates the interface between an air sac and a capillary blood vessel. In the original design reported in 2010, human alveolar epithelial cells line one side of a thin, flexible membrane while lung capillary endothelial cells line the other. Air flows over the epithelial side, culture medium mimicking blood flows past the endothelial side, and a vacuum applied to side chambers rhythmically stretches the membrane to simulate breathing.5PubMed Central. Reconstituting organ-level lung functions on a chip
That breathing motion turns out to matter enormously. When researchers exposed the chip to silica nanoparticles, the cyclic stretching amplified both the toxic response and the rate at which particles crossed the barrier into the “blood” channel. The same effect was confirmed in whole mouse lungs, suggesting the chip was capturing a genuine physiological phenomenon that static cultures miss entirely.5PubMed Central. Reconstituting organ-level lung functions on a chip A later iteration of the device went further, reconstituting the three-dimensional microarchitecture and dynamic mechanical activity of the alveolar-capillary interface to study whole-organ-level responses to bacteria and inflammatory signals.6PubMed Central. A human breathing lung-on-a-chip
Some chips now come with built-in sensors. One lung-cancer-on-a-chip platform, for example, integrates biosensors that continuously measure the electrical resistance across the cell barrier, giving researchers a real-time readout of how a drug candidate is affecting tissue integrity without having to stop the experiment and take samples.7Biochemical Engineering Journal. A lung cancer-on-chip platform with integrated biosensors for physiological monitoring and toxicity assessment
Lung Organoids Grown from Stem Cells
Organoids take a different approach: instead of engineering a device and seeding cells onto it, researchers coax stem cells to self-organize into tiny, three-dimensional clusters that recapitulate features of lung tissue. Human induced pluripotent stem cells can be guided through a series of chemical signals to become alveolar epithelial cells and then form alveolar organoids in culture.8PubMed. A method of generating alveolar organoids using human pluripotent stem cells Because the starting cells can come from a patient’s own skin or blood sample, the resulting organoids carry that patient’s complete genetic makeup, including any disease-causing mutations.
One recent advance is the development of matrix-free lung organoids derived from iPSCs. Traditional organoid protocols require an animal-derived gel matrix, which introduces variability and can interfere with certain experiments. A matrix-free method generates human lung organoids that can be used to investigate lung damage in a way that is closer to the patient’s own biology and avoids reliance on animal models.9PubMed Central. Matrix-free human lung organoids derived from induced pluripotent stem cells to model lung injury
Organoids have proven especially useful in cancer research. In non-small cell lung cancer, 3D organoid co-cultures allow researchers to seed immune cells alongside tumor cells and observe how they interact inside a structure that mimics the tumor microenvironment. These platforms are enabling new studies of macrophage-targeted immunotherapies that would be nearly impossible to study in a flat dish.10eLife. The role of macrophages in non-small cell lung cancer and advancements in 3D co-cultures
Bioprinting Lung Tissue
Bioprinting brings the precision of 3D printing to living tissue. The general idea is to load cells and biologically active molecules into a printable gel, called a bioink, and then deposit them layer by layer to recreate the architecture of airways or alveoli.11PubMed Central. 3D bioprinting of the airways and lungs for applications in tissue engineering and in vitro models The challenge is finding a bioink that is stiff enough to hold its shape during printing but soft enough to let cells survive, grow, and connect with their neighbors.
Several bioink strategies are being explored. One uses a composite of methacrylated mucin and hyaluronic acid that can be cross-linked with visible light at 405 nanometers, gentle enough to avoid damaging cells. The resulting scaffolds are porous, which helps nutrients diffuse inward and lets cells migrate through the structure.12PubMed. 3D Bioprinting with Visible Light Cross-Linkable Mucin-Hyaluronic Acid Composite Bioink for Lung Tissue Engineering Another approach reinforces the bioink with extracellular matrix proteins, enabling the printing of human airway constructs composed of regionally specified progenitor cells and smooth muscle cells. In that study, the printed airway lumens remained open with viable cells for a full month, and cells differentiated into mature epithelial types found in native airways.13PubMed Central. Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue
Bioprinting is still far from producing a transplantable lung, but it is already useful for creating standardized tissue models for drug screening and toxicology. The ability to precisely position different cell types within a construct means researchers can study, for instance, how an airway lining and its underlying smooth muscle respond to a drug simultaneously.
Decellularized Lung Scaffolds
Perhaps the most ambitious approach to 3D lung engineering involves taking an actual donor lung, stripping away every cell, and using the remaining extracellular matrix as a natural scaffold. The idea is that this ghost organ retains the intricate branching architecture, basement membranes, and mechanical properties that no synthetic material can yet replicate. Researchers then reseed the scaffold with new cells, ideally derived from the intended recipient, to rebuild a functional organ.14PubMed Central. Revascularization of decellularized lung scaffolds: principles and progress
In rodent studies, bioengineered lungs recellularized with airway and vascular cells have been implanted and shown to exchange gas for up to 14 days.14PubMed Central. Revascularization of decellularized lung scaffolds: principles and progress The results are encouraging but far from clinical readiness. One persistent problem is that the decellularization process changes the ratio of remaining matrix proteins: collagen is retained while certain glycoproteins are lost. That shift makes it harder for new cells to attach and spread evenly. In one study, reseeding with mouse embryonic stem cells was initially inefficient, with only about 7% of cells successfully engrafting. Pre-treating the scaffold with conditioned media containing laminin more than doubled that efficiency and improved how uniformly cells distributed throughout the organ.15PubMed Central. Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells
Modeling Infectious Disease
The COVID-19 pandemic thrust lung-on-a-chip technology into the spotlight. Researchers created a human alveolar chip by co-culturing alveolar epithelial cells, microvascular endothelial cells, and circulating immune cells under fluid flow, then infected the system with SARS-CoV-2. The chip revealed that the epithelium was far more susceptible to infection than the endothelium, and that the two cell types activated distinct molecular pathways at three days post-infection. Immune cell recruitment drove endothelial detachment and a surge of inflammatory signaling molecules, mirroring the cytokine-driven lung injury seen in severe COVID patients. When remdesivir was added, it inhibited viral replication and reduced barrier disruption on the chip.16PubMed Central. Biomimetic Human Disease Model of SARS-CoV-2-Induced Lung Injury and Immune Responses on Organ Chip System
This kind of experiment would be extremely difficult in traditional cell cultures, which lack immune cells and fluid flow, and ethically fraught in animal models where the virus may behave differently. Lung-on-a-chip platforms are now being developed more broadly for evaluating antiviral drugs against a range of respiratory viruses, from influenza to respiratory syncytial virus.17PubMed Central. Biomimetic lung-on-a-chip to model virus infection and drug evaluation
Personalized Medicine and Genetic Disease
Because organoids can be grown from a specific patient’s cells, they open the door to genuinely personalized drug screening. Cystic fibrosis is a prime example. In one study, researchers generated iPSCs from a cystic fibrosis patient carrying a specific nonsense mutation in the CFTR gene, then grew those cells into 3D airway organoids that displayed hallmark CF features, including the expected mix of airway epithelial cells, basal cells, and goblet cells. Using single-cell analysis, the team identified three drugs, nintedanib, N-acetylcysteine, and losartan, that reduced a key inflammatory pathway in the organoids.18PubMed. Multimodal single-cell transcriptomics with patient-specific iPSC-derived airway organoids as a drug screening approach for cystic fibrosis with nonsense mutations
The significance here is not just that the model works but that it can be tailored to each patient’s mutation. Cystic fibrosis involves more than 2,000 known CFTR mutations, and existing therapies only address a fraction of them. A patient-specific organoid could, in principle, let clinicians test which drugs actually help a given individual before prescribing anything. That vision is still being refined, but the underlying technology is already functional in research settings.
Inhalation Toxicology and E-Cigarette Research
Three-dimensional lung models are particularly well suited to studying inhaled substances because you can deliver aerosols directly to an exposed cell surface rather than dissolving them in liquid. One team built a ventilated 3D-printed artificial lung system with anatomically accurate branching airways and used it to study how particles of different sizes deposit during simulated breathing. They observed a U-shaped deposition pattern: very tiny particles (around 10 nanometers) deposited at higher rates because of random Brownian motion, and very large particles (around 10 micrometers) deposited heavily due to gravity and inertia. Intermediate-sized particles largely sailed through.19Environmental Science & Technology. A Ventilated Three-Dimensional Artificial Lung System for Human Inhalation Exposure Studies
For e-cigarettes specifically, a validated ALI model using Calu-3 cells cultured for 28 days was exposed to aerosols from three flavored e-liquids and compared to traditional cigarette smoke. The model confirmed that hazelnut and cinnamon e-liquid aerosols significantly disrupted the cell barrier and caused cell damage relative to air-only controls, while vanilla tobacco did not show the same effect. Traditional cigarette smoke remained the most damaging. The model tracked damage using electrical resistance measurements and markers of cell death, providing a more physiologically relevant picture than simply soaking cells in dissolved e-liquid.20PubMed Central. Toxicological assessment of E-cigarette flavored E-liquids aerosols using Calu-3 cells: A 3D lung model approach
The Vascularization Problem
The lung is one of the most densely vascularized organs in the body. Every air sac is wrapped in a mesh of capillaries so fine that red blood cells squeeze through single file. Recreating that vascular network in an engineered tissue remains the single biggest technical barrier to building anything approaching a functional replacement lung.
Progress is being made on multiple fronts. Using advanced hydrogel printing, researchers have fabricated soft structures with complex internal vascular networks that are stiff enough to withstand perfusion without bursting and can simulate breathing. In one demonstration, red blood cells flowing through printed “blood vessels” were able to pick up oxygen from adjacent “alveoli,” closely mimicking native gas exchange. When implanted in mice, the vascularized tissues survived, indicating that the printed vessels could transport nutrients to cells.21Smart Materials in Medicine. Recent progress of 3D printed vascularized tissues and organs
Decellularized scaffold approaches face a related challenge. When a team transplanted a bioengineered mouse lung seeded with human endothelial cells, the cells formed patent capillary lumens, but some were too narrow for red blood cells to pass through. After transplantation and about ten minutes of blood circulation, histology showed blood had reached the most peripheral capillaries, with red blood cells sitting inside lumens formed by the transplanted endothelial cells. Only minor hemorrhage was observed.22Scientific Reports. Orthotopic transplantation of the bioengineered lung using a mouse-scale perfusion-based bioreactor and human primary endothelial cells Ten minutes of perfusion is worlds away from a permanent transplant, but the fact that engineered capillaries could circulate real blood at all marks meaningful progress.
Regulatory Shifts and the FDA Modernization Act 2.0
Until recently, U.S. law effectively required animal testing before a new drug could enter human clinical trials. The FDA Modernization Act 2.0, signed in late 2022, changed that. The law now allows drug developers to use alternatives to animal testing, including organ-on-chip devices, organoids, computer simulations, and other advanced in vitro models, to satisfy preclinical safety requirements.23PubMed. FDA Modernization Act 2.0: An insight from nondeveloping country This does not ban animal testing, but it removes the legal mandate that had kept it as the default pathway.
The practical impact is already visible. Pharmaceutical companies and regulators are increasingly interested in data generated by organ-on-chip platforms, and the FDA has accepted chip-based data in several investigational new drug applications. At the same time, researchers and regulators have flagged outstanding issues around standardization: different labs build chips in different ways, use different cell sources, and measure different endpoints, making it hard to compare results across studies or establish universal benchmarks.24Med. Organoids and organs-on-chips: From fundamental research to regulatory perspectives and pharmaceutical applications Solving that standardization problem is probably the most important near-term challenge for bringing these models into routine regulatory use.
Chronic Lung Disease on a Chip
Idiopathic pulmonary fibrosis is one of the most difficult lung diseases to study and treat. The scarring that characterizes IPF progresses relentlessly, and only two approved drugs slow it modestly. Animal models of fibrosis use chemical injury to trigger scarring, which does not perfectly reflect the slow, idiopathic onset of the human disease. Lung-on-a-chip systems and other 3D models are now being developed to recapitulate the mechanical stiffening, abnormal cell signaling, and matrix remodeling that define IPF, offering a platform where candidate anti-fibrotic therapies can be tested against human cells in a more realistic mechanical environment.25PubMed Central. In Vitro Modeling of Idiopathic Pulmonary Fibrosis: Lung-on-a-Chip Systems and Other 3D Cultures
The mechanical dimension is especially relevant in fibrosis. As scar tissue builds up, the lung becomes stiffer, and that stiffness itself feeds back to promote more scarring. Capturing that feedback loop requires a model that can apply and vary mechanical forces, something flat cultures simply cannot do. Chip-based systems with tunable membrane stiffness or stretch patterns are well positioned for this kind of work, though the field is still relatively young compared to chip-based infection or toxicology models.
Where the Field Is Headed
Multi-organ chip systems, which connect a lung chip to a liver chip, a heart chip, or a kidney chip through shared fluid channels, are being developed to study how an inhaled drug affects not just the lungs but the whole body. If a compound is absorbed through the lung lining, metabolized by the liver chip, and then causes toxicity in the heart chip, that information could prevent a dangerous drug from ever reaching human trials. These systems are still in early development, but the modular nature of organ-on-chip technology makes the plumbing conceptually straightforward even if the biology remains tricky.
At the tissue-engineering end, the dream remains a transplantable bioengineered lung. The gap between a scaffold that exchanges gas for two weeks in a rodent and a permanent human organ replacement is vast, involving challenges in vascularization, immune compatibility, long-term cell survival, and scaling up from a mouse-sized organ to a human-sized one. Most researchers in the field are candid that a transplantable bioengineered human lung is likely decades away. In the meantime, the near-term payoff of 3D lung models sits squarely in drug development, toxicology, and personalized medicine, areas where even a small, imperfect model of human tissue can yield information that no animal experiment or flat dish ever could.