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

Abstract

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 in vitro 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.

Keywords: 3D culture; ACE2; COVID-19; MatriWell; Respiratory diseases; SARS-CoV-2.

Graphical abstract

Fig. 1

Morphological analysis and development of BEAS-CTL and BEAS-ACE2 spheroids and 2D cells. (A) Scaffold-free spheroid-producing technique using agarose micro-molds. (B) Bright-field images of the spheroids on days 1, 7, and 14. (C) Spheroids historesin assay. Comparison of spheroids’ (D) diameter and (E) volume. (F) Bright-field images of 2D cultures of BEAS-CTL and BEAS-ACE2 cells. (G) Area measurement of 2D cultures of BEAS-CTL and BEAS-ACE2 cells. Scale bar: 50-100 µm. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. n = 25-50.

Fig. 2

BEAS-CTL and BEAS-ACE2 spheroids viability over time. The Viability over time was quantified using the trypan blue staining. *p ≤ 0.05, n = 3.

Fig. 3

Viral load quantification and immunofluorescence assay of BEAS-CTL and BEAS-ACE2 spheroids. (A) Spheroid intracellular viral concentration. (B) Immunofluorescence microscopy images of the spheroids with nucleus (Hoechst), ACE2, and Spike protein staining. Fluorescence quantification of (C) Spike protein and (D) ACE2 expression. (E) Pearson correlation comparing ACE2 and Spike protein expression. Scale bar: 50 µm. *p ≤ 0.05; **p ≤ 0.01. n = 3-5

Fig. 4

SARS-CoV-2 infection of the 3D models. (A) SARS-CoV-2 infection of spheroids and MatriWells. (B) Comparison of intracellular viral load in BEAS-CTL spheroids and MatriWells. (C) Comparison of intracellular viral load in BEAS-ACE2 spheroids and MatriWells. ****p ≤ 0.0001, *p≤0.05. n = 5-15

Fig. 5

MatriWells representative scheme and SARS-CoV-2 kinetics in BEAS-ACE2 MatriWells and HUVEC cells. (A) Pulmonary ALI creation using the MatriWells, BEAS-ACE2 and HUVEC cells. (B) Intracellular viral load in BEAS-ACE2 MatriWells. (C) Number of viral particles per mL(PFU[plaque-forming units]/mL) in HUVEC's supernatant. (D) Intracellular viral load in HUVEC cells. *p ≤ 0.05; ***p ≤ 0.001. n = 12.