In Vitro Lung Models for Drug Toxicity Testing

The National Institutes of Health (NIH) has announced the end of funding for grant proposals solely relying on animal testing. From July 2025, all new NIH funding opportunities moving forward should incorporate language in consideration of New Approach Methodologies (NAMs). This follows the April announcement of the Food and Drug Administration (FDA) to phase out the animal testing requirement for monoclonal antibodies and other drugs.

It’s time for alternative models: models that allow extrapolation back to the human exposures and bridge the gap between current 2D in vitro approaches and human clinical trials.

 

Comparison of In Vitro Lung Models: 2D, 3D, and Organ-on-a-Chip Approaches

In vitro lung models have evolved significantly, ranging from simple 2D monolayers to complex microengineered systems. Each format offers distinct advantages and trade-offs in terms of complexity, cost, scalability, and physiological representativity.

2D monolayer cultures, often composed of immortalized or primary epithelial cells, remain the most widely used format, particularly in regulatory drug toxicity assays. These models are relatively inexpensive and allow for high-throughput screening, but their limited structural complexity makes them less representative of in vivo lung architecture. Nonetheless, 2D systems, the only OECD-regulated model, have contributed substantially to our understanding of cell-specific responses, particularly in screening for cytotoxicity and barrier integrity.

Advancing from flat monolayers, 3D models, such as air-liquid interface (ALI) cultures, co-cultures, and organoids, offer greater physiological fidelity. ALI systems allow epithelial cells to differentiate into multiple subtypes, including goblet and ciliated cells, thereby recreating key functions like mucociliary clearance and surfactant production (Figure 1b). Co-cultures introduce immune or endothelial cells to further approximate the lung microenvironment (Figure 1c). Organoids, derived from stem or progenitor cells, self-organize into spherical structures containing multiple cell types from specific lung regions (Figure 1e). They hold promise for long-term toxicity assays and disease modeling but face challenges in standardization, scalability, and full maturation.

 

Figure 1. Lung cell model systems. a) Submerged monocultures – tend to be an epithelial cell layer with liquid above and below the cells. b) An air-liquid interface (ALI) culture. A layer of cells grown on a transwell insert with medium on the basal side and air on the apical side. c) co-culture models, which are also cultured at the ALI, but instead of a monolayer of epithelial cells, they also tend to contain immune cells or endothelial cells. d) Commercial models – contain a bigger diversity of cells and are delivered to the end user ready to be used. e) Organoids – can be made up of various cells with a round structure. f) (Bio)Scaffolds – help advance the cell culture models by giving the cells a structure to grow upon. g) Lung-on-a-chip models – aim to replicate the lung, including fluidics, at a much smaller scale. h) 3D Bioprinting – can “print” specific cells layer upon layer to mimic a specific area of the lung. i) Bioreactors – allow the addition of both fluid-flow and breathing mechanics to a culture that was previously static. Source: Moura JA et al. Alternative lung cell model systems for toxicology testing strategies: Current knowledge and future outlook. Semin Cell Dev Biol. 2023 Sep 30;147:70-82.

 

Complementing these models, scaffold-based systems (Figure 1f) and 3D bioprinting allow precise control over tissue geometry and composition. While still emerging, bioprinted constructs (Figure 1h) have successfully reproduced airway and alveolar features, offering a promising platform for drug toxicity testing. 

Perhaps the most cutting-edge approach lies in lung-on-a-chip (LoC) technologies (Figure 1g). These microfluidic devices combine epithelial and endothelial cells across a flexible membrane, with dynamic flow and mechanical stretch to simulate breathing. They have demonstrated exceptional potential in replicating alveolar-capillary interfaces, enabling real-time monitoring of responses to drugs, pathogens, and airborne particles. Their modularity also supports multi-organ integrations, allowing for assessment of systemic effects in interconnected platforms.

LoC is being exploited in major European initiatives, such as the UNLOOC, which aims to develop, optimize, and validate organ-on-a-chip systems for diverse organs, including the lung. As proud partners, BeCytes Biotechnologies provides lung cells to the consortium.

 

Applications of Lung In Vitro Models in Preclinical Drug Development

In vitro lung systems are increasingly vital during the early stages of drug toxicity testing. By enabling mechanistic insights and toxicity profiling, these models can reduce late-stage attrition and guide dosage optimization: 

• Cytotoxicity and barrier integrity assays are routinely performed on 2D and ALI cultures to screen for harmful compounds. 2D cultures serve for yes/no assessments, but fall short in capturing the complex, dynamic responses that 3D models can reveal.
• Drug delivery and absorption studies benefit from models that replicate the air-liquid interface, as they simulate the real-world scenario of aerosolized drugs contacting the epithelial surface. Some organ-on-a-chip devices go further, incorporating fluid flow and cellular breathing motions, which influence particle deposition and distribution.
• Disease-specific applications are particularly promising. Using patient-derived cells, we can create organoids or chips that mimic pathological conditions like cystic fibrosis, asthma, idiopathic pulmonary fibrosis, or lung cancer. This allows for personalized testing of drug candidates and investigation of disease mechanisms under near-physiological conditions.

 

 

• Repeated dose and chronic exposure assessments, which are difficult to implement in vivo due to ethical and logistical constraints, are now feasible with long-lived 3D cultures and organoids. These systems maintain viability for weeks, allowing for accumulation studies and detection of delayed toxic effects.

In vitro pulmonary models for drug development have progressed from simplistic cell layers to sophisticated engineered tissues capable of mimicking key aspects of human respiratory physiology. 

At BeCytes, we are glad to contribute to this shift away from animal testing by providing primary human cells and tissue sourcing services. We are dedicated to accelerating scientific advancements while ensuring excellence at every step of the process.

 

 

We have plenty of experience in isolating cells and procuring tissue for lung in vitro models for preclinical drug testing. If you are looking for a reliable partner, contact us!

 

References

Moura JA, Meldrum K, Doak SH, Clift MJD. Alternative lung cell model systems for toxicology testing strategies: Current knowledge and future outlook. Semin Cell Dev Biol. 2023 Sep 30;147:70-82. doi: 10.1016/j.semcdb.2022.12.006