TABLE 2.

Main challenges associated with the four different in vitro culture systems for modelling lung environment in health and disease

PCLS	Organoids	Lung ECM-derived hydrogels	Lung-on-chip
Patient-to-patient variations	Heterogeneity in number and sizes (due to intra- and inter-patient variations and various culture modalities)	Patient-to-patient variations influencing ECM hydrogel properties	Challenges associated with incorporation of cells (only for closed lung-on-chip systems)
Heterogeneity in the diseased regions, resulting in intra- and inter-slice differences	Closed architecture with limited access to the lumen, which is filled with liquid instead of air	Heterogeneity in the diseased regions and in decellularisation methods	Restriction of the morphogenesis to predefined geometry
Absence of easy air–liquid interface setting	Presence of necrotic cores	Macroscopic architecture differing from native lung	Non-permissive environment that cannot be remodelled
Need for fresh starting material that limits lifespan of various cells and the tissue in vitro	Difficult to obtain fully differentiated lung cell types	Pepsin removal currently not possible	Non-physiological stiffness of the device
Snapshot of the cell populations in the tissue and absence of access to infiltrating cells	Lack of vascularisation and difficult to introduce perfusion or immune cells	Incorporation of cells is challenging	Challenges associated with translation of observations from microscale to in vivo systems
Difficult to preserve arteriole morphology and function	Limited inclusion of mechanical forces associated with breathing	Limited inclusion of mechanical forces associated with breathing	For non-commercialised lung-on-chips: rather long production time of the microfluidic devices with a small throughput
Cellular behaviour likely impacted by processing and agarose embedding	Limitations in hydrogel types to develop organoid culture systems	Lack of vascularisation and difficult to introduce perfusion

PCLS: precision-cut lung slice; ECM: extracellular matrix.