Characterization of 3D human pulmonary epithelial model morphology and oxygen status under normoxia and hypoxia

Infection generates localized hypoxia in affected tissue, inducing cellular survival responses and modulating inflammatory processes. Consideration of oxygen status as a parameter in in vitro infection research is therefore vital to the generation of physiologically relevant data within the 3R context. In this study, we characterize the culture morphology and oxygenation of liquid-liquid interface (LLI) permanent bronchial epithelial (Calu-3), classical air-liquid interface (cALI) Calu-3, and cALI human primary bronchial epithelial cell (hBEC) models under the normoxic conditions within standard incubators, commonly employed in in vitro work. We compare the normoxic state of these models to their hypoxic state to assess changes in the airway epithelial environment in response to oxygen deprivation, and the extent to which select hypoxia responses can be observed at the molecular level. Additional juxtapositions are drawn between Calu-3 LLI and cALI models and Calu-3 conventional monolayer (CM) and inverted air-liquid interface (iALI) models, due to their relevance for basic and specialized research, respectively. Epithelial complexity was observed to vary amongst the filter-based models, and all models were found to exhibit characteristic extracellular oxygen depletion patterns under normoxia. Importantly, the extracellular oxygen contents of Calu-3 LLI, cALI, and CM models significantly decreased during normoxic incubation. Specific hypoxia responses through stabilization of HIF-1α, HIF-2α, and/or HIF-3α and alteration of ACE2 protein levels differed in response to both culture format and cell type. Therefore, while all models examined provide valuable opportunities for in vitro exploration, variation in their morphological, physiological, and molecular characteristics necessitates careful consideration during experimental design.