Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection.

COVID-19, along with most respiratory diseases in the medical field, demonstrates significant ability to take its toll on global population. There is a particular difficulty in studying these conditions, which stems especially from the short supply of models for detailed investigation, the specific therapeutic knowledge required for disease scrutinization and the occasional need of BSL-3 [Biosafety Level 3] laboratories for research. Based on this, the process of drug development is hampered to a great extent. In the scenario of COVID-19, this difficulty is even more substantial on account of the current undefinition regarding the exact role of the ACE2 [Angiotensin-converting enzyme 2] receptor upon SARS-CoV-2 kinetics in human cells and the great level of demand in the investigation process of ACE2, which usually requires the laborious and ethically complicated usage of transgenic animal models overexpressing the receptor. Moreover, the rapid progression of the aforementioned diseases, especially COVID-19, poses a crucial necessity for adequate therapeutic solutions emergence. In this context, the work herein presented introduces a groundbreaking set of 3D models, namely spheroids and MatriWell cell culture inserts, whose remarkable ability to mimic the in vivo environment makes them highly suitable for respiratory diseases investigation, particularly SARS-CoV-2 infection. Using MatriWells, we developed an innovative platform for COVID-19 research: a pulmonary air-liquid interface [ALI] associated with endothelial (HUVEC) cells. Infection studies revealed that pulmonary (BEAS-2B) cells in the ALI reached peak viral load at 24h and endothelial cells, at 48h, demonstrating lung viral replication and subsequent hematogenous dissemination, which provides us with a unique and realistic framework for studying COVID-19. Simultaneously, the spheroids were used to address the understudied ACE2 receptor, aiming at a pronounced process of COVID-19 investigation. ACE2 expression not only increased spheroid diameter by 20% (p<0.001) and volume by 60% (p≤0.0001) but also led to a remarkable 640-fold increase in intracellular viral load (p≤0.01). The previously mentioned finding supports ACE2 as a potential target for COVID-19 treatment. Lastly, we observed a higher viral load in the MatriWells compared to spheroids (150-fold, p<0.0001), suggesting the MatriWells as a more appropriate approach for COVID-19 investigation. By establishing an advanced method for respiratory tract conditions research, this work paves the way toward an efficacious process of drug development, contributing to a change in the course of respiratory diseases such as COVID-19.