The lung in crisis: fighting back against pathogens, infections and inflammation with predictive in vitro lung models that accelerate drug discovery

The human lung is in crisis

Our lungs represent the only internal organ that is directly subject to the environment and therefore face a relentless battle against foreign matter. For centuries, they have been assaulted by enemies – both natural and synthetic. Biological intruders such as viruses, bacteria and fungi constantly test defences that have evolved over thousands of years, whilst newer, stranger particles now enter the lung on an increasingly frequent basis, originating from burning fuels, disintegrating plastics and harmful chemicals made by our own human hands.

As a result, lung diseases have become one of the biggest causes of death; with chronic obstructive pulmonary disease (COPD), lower respiratory infections and lung cancer making up three of the top ten causes worldwide (World Health Organization (WHO), 2020). Unfortunately, the recent COVID-19 pandemic has further exacerbated this global crisis. As of March 2022, there has been almost 450 million confirmed cases of COVID-19 and 6 million deaths as reported by the WHO (WHO, 2022).

Figure 1. Leading causes of death globally – WHO Global Health Estimates.

Luckily for us, the resilient lung is well defended against encountered dangers. Specialised cells called Goblet cells produce a layer of gel-like mucus which coats the airway epithelium. Any larger particulate matter that enters the airway will become entrapped in the mucus. Then, via the “beat” of the cilia – a brush-like structure formed by ~50% of airway cells – the mucus is moved back up the airway and into the digestive system to be destroyed. This cycling of mucus – or mucociliary clearance – stops many, but not all, attacks to the lung.

Figure 2. Bronchial epithelium. Tissue generated using the CN Bio PhysioMimix® OOC and Barrier (MPS-T12) Plates. Ciliated cells (yellow), mucus (green), actin cytoskeleton (red) and nuclei (blue) are indicated.

Cunning invaders that pass through frontline defences to reach the alveoli are only 0.2 μm away from reaching the bloodstream. However, the first immune sentinel of the lung – the alveolar macrophage – stands in their way. Alveolar macrophages patrol the internal portion of the alveoli and control the switch between anti-inflammatory tolerance and inflammatory attack response to foreign bodies. Intense inflammatory responses result in infiltration of circulating immune cells from the systemic blood whose aim is to destroy the invader but, unfortunately, this often causes damage to the alveoli too. In short, the wrong pathogen combined with inflammatory signalling from alveolar macrophages can represent the difference between a normal day and a trip to the emergency room.

This functional diversity, orchestrated by a multitude of pulmonary and immune cells, has proven challenging for scientists to reproduce in the laboratory. Traditional in vitro models range from simple 2D cell line cultures to 3D spheroids and scaffold models. Despite significant efforts and much improvement over the years, standard approaches fail to replicate many of the lung’s complex interactions

To overcome these shortfalls, animal models are widely used for lung research, despite differences in physiology and biology compared to human lung (Muñoz-Fontela et al., 2020; Sarkar & Heise, 2019). This disparity manifests in comparatively high drug attrition rates, with only 3% of pulmonary therapeutics reaching the market compared to 6-14% of therapeutics for other diseases (Barnes et al., 2015). Ex vivo studies, which utilise isolated, perfused lungs or tissue slices from human, represent an option to reduce animal experimentation and bring more physiological relevance to studies (Hocke et al., 2017; Viana et al., 2021); however, whole human lung tissues can be difficult to obtain, therefore precision cut lung slices (PCLS) provide a more cost-effective alternative.

The pros and cons of traditional approaches are summarised in more detail in Table 1 below, but the lack of easily accessible, cost-effective, human-representative preclinical models has resulted in preclinical pipeline blocks and shocking drug attrition rates.

Model	Advantages	Disadvantages
2D insert cell culture	Low cost High throughput Air-liquid interface (ALI) capacity Ability to coculture cell types	Often cell lines – not as representative as primary human cells Lack of 3D structure formation Long timeframe differentiation protocols
Spheroid/organoid culture	3D integrated tissue formed Multiple cell types Use of stem cells / primary human cells ALI capacity	Expensive and long timeframe experiments (stem cells) Bulk sphere of cells not physiologically relevant for lung
Scaffold cultures	Scaffolds allow formation of lung relevant 3D architecture Cell-ECM interaction	Currently challenging to monitor cells in scaffolds Post culture analysis is difficult
Ex vivo	Full organ with heterogeneous cell population Human ex vivo lung is fully representative Lungs can be perfused post isolation Precision cut lung slices are available	Difficult to obtain human tissue. Small animal lung is not representative of human lung physiology Operates in isolation from the full system
In vivo	Ability to understand system biology interactions Full working organs with heterogeneous cell population Non-human primates have similar physiology to human lung	Ethically questionable Expensive and time consuming Low availability of non-human primates Small animal lung physiology is different to human Controlling studies at the cellular level and investigating molecular mechanisms and/or genetic drivers is difficult

Table 1. The pros and cons of traditional preclinical lung models

With the elevated risk of new pathogens causing future global pandemics, plus the air that we breathe becoming pollutant laden from things we take for granted – such as vehicles, gas central heating, paints, cleaning fluids, solvents and even farms – more rapid production of vaccines and therapeutics will be required. After the COVID-19 pandemic, organisations have begun to understand this requirement, for example the Bill Gates Foundation and Wellcome Trust recently donated $300 million dollars to the Coalition for Epidemic Preparedness Innovations (CEPI) with the aim to cut the time required to develop new vaccines. The same preparedness should be applied to the preclinical pipeline, whereby the effect of new pathogens, or irritants can be rapidly understood, therapeutics designed and tested to predict their efficacy and safety in human before final release to clinical trials.

Figure 3. Alveolar epithelium. Tissue generated using the CN Bio PhysioMimix® OOC and Barrier (MPS-T12) Plates. ATII cells (yellow), actin cytoskeleton (magenta) and nuclei (blue) are indicated.

To meet this need, cost-effective, human-relevant models that faithfully recapitulate both healthy and diseased lung biology are urgently required. Lung-on-a-chip models (LOAC), alternatively known as lung microphysiological systems (MPS), have been in development over the last decade. Encouragingly, they have been shown to mimic aspects of the human lung better than other in vitro, thereby representing a potential solution.

A wide variety of LOAC models are now available, ranging from single-use microscale chips to insert-based multi-well plate systems that provide an easier way to incorporate immune cells and apply physiologically-relevant infection parameters (Artzy-Schnirman et al., 2019; Huh et al., 2010; Stucki et al., 2018; Zamprogno et al., 2021). LOAC models contain a combination of integrated microfluidics, scaffolds and stretchable membranes that expose cells to the shear stresses of the human lung, encouraging them to form microtissues that accurately mimic the human lung’s pathophysiology, as well as integrated sensors to monitor the tissues in real time. The more flexible solutions can be adapted to increase model complexity, via the co-culture of a wide range of cells, to address more specific research questions (Barkal et al., 2017; Humayun et al., 2018; Sellgren et al., 2014).

Model	Advantages	Disadvantage
Organ-on-a-chip (OOC) or microphysiological systems (MPS)	3D integrated tissues form Use of human primary / stem cells with capability to coculture multiple cell types Use of scaffolds ALI capability Perfused media or air provides shear stress for further differentiation potential Flexible membranes mimic movement of lung during breathing Integrated sensors for impedance, barrier integrity, pH, microscopy	Lower throughput and more expensive than standard cell culture Adoption barriers exist for OOC systems featuring non-standard cell culture designs Closed OOC systems are more difficult to infect and monitor inflammatory responses than open systems Some OOC solutions do not offer recirculating media/cannot incorporate immune cells into the model

Table 2. The pros and cons of next generation LOAC.

Their ability to recreate physiologically relevant human lung tissue and monitor tissues in real time, means that LOAC models are well suited to studies that investigate infection and inflammatory responses to foreign matter (Benam et al., 2020; Deinhardt-Emmer et al., 2020; Zhang et al., 2020). Open-well format systems are particularly useful as test matter can be delivered into to the system as aerosols or small volumes at the ALI, and samples for analysis can be easily removed over time for longitudinal studies. The potential efficacy and safety of therapies in development can be tested via addition to the system apically at the ALI to simulate the dosing of inhaled medications, or basolaterally to mimic oral or intravenous therapies, whilst systemic effects can be elucidated by interconnecting relevant MPS organs together to form multi-organ systems (Chen et al., 2017; Edington et al., 2018). A summary of pros, cons and considerations when choosing an OOC solution can be found in Table 2.

Having read thus far, I hope you would agree that LOAC models offer much promise, but can they save the lung from crisis? Could they represent the missing piece of the preclinical puzzle? What if the complimentary use of LOAC models alongside traditional approaches eliminates our preclinical bottlenecks? Only time and further research will tell, but for now more you can learn more about LOAC models and their potential applications, at our next webinar:

A breath of fresh air: novel approaches to human lung disease modelling for accelerated drug discovery.

Presented by Professor Wojciech Chrzanowski, Faculty of Medicine and Health, University of Sydney & Dr Emily Richardson, Lead Scientist – Assay Development, CN Bio on March 29^th. We look forward to seeing you there!

Follow the links to learn more about CN Bio’s PhysioMimix OOC Systems and Lung-on-a-chip barrier models

AUTHOR
Dr Emily Richardson
Lead Scientist Assay Development – CN Bio

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