Despite the availability of safe and effective vaccines against COVID-19, new variants of concern (VOC) such as Delta and Omicron are emerging. In these surges, governments and institutions around the world urgently need ways to rapidly respond to outbreaks of new VOCs or other infectious respiratory diseases with pandemic potential.
In this context, tissue-engineered models serve as valuable tools for immediate preclinical and translational research regarding therapeutics and prophylactics. Models faithfully mimicking human respiratory mucosa and submucosa could support research and development regarding COVID-19 and other diseases caused by pathogens entering the body by the respiratory route.
Human bronchial fibroblasts (HBFs) isolated from cryo-biopsies were combined with human umbilical vein endothelial cells (HUVECs) inside 3D fibrin hydrogels. Primary human bronchial epithelial cells (HBEs) were seeded on top of the gels, which were then grown on transwell inserts for one week as submerged culture followed by 4 weeks of air-liquid interface culture. The models were fixed and CD31/PECAM-1 and the SARS-CoV-2 entry receptor hACE2 were analyzed by 2-Photon Laser Scanning Microscopy (2P-LSM) and immunohistochemistry, respectively. Furthermore, cultures were examined by Scanning Electron Microscopy (SEM).
SARS-CoV-2 viruses were cultured in Calu-3 cells, isolated, and added from either the apical side, the basal, or both. Cultures were fixed 8 days post-treatment and immunostained for the viral Nucleocapsid (N) protein to visualize the infection. 2D monocultures of HBFs and HUVECs were also infected and analyzed for spike (S) and N protein expression by a newly developed in-cell-ELISA1.
Additionally, a clinical isolate of the VOC Delta was cultured in Calu-3 cells to prepare virus particles that were inactivated by ultraviolet light (UV) irradiation (“UV-Delta”). UV-Delta was added to the aforementioned airway 3D models, which were incubated for 4, 24, or 48 hours, fixed, and examined by histological periodic acid Schiff´s (PAS) reaction and SEM.
Similar to native tissues, histology of the airway tri-cultures showed high expression of the SARS-CoV-2 receptor hACE2 mainly in the epithelium layer. The 2P-LSM 3D stacks of the CD31/PECAM-1 staining showed the HUVECs arranged in a network of capillary-like structures, while the SEM confirmed the presence of a fully developed ciliated epithelium on the apical side of the model.
While the icELISA detection in 2D monocultures verified that both HUVECs and HBF are in principle susceptible to SARS-CoV-2 infection, the N protein immunostaining of tri-cultures revealed that complex models are only permissive to SARS-CoV-2 when exposed to the virus from the epithelium side. The PAS reaction showed a weakening and thinning of the epithelial barrier when exposed to UV-Delta, while the SEM analysis revealed an increase in ciliation of the epithelium after 24 and 48 hours.
Our airway tri-cultures represent a complex model for research regarding the pathogenesis of SARS-CoV-2 and therapeutics against COVID-19. The treatment of the tri-cultures with inactivated viral particles from the VOC Delta induces an increase in ciliation, a counterintuitive effect that differs from the findings with live SARS-CoV-2 and other coronaviruses.
- Schoeler, L. et al., Front. Immunol. 11, 573526 (2020)."