Establishing human respiratory epithelial organoid culture systems for modeling SARS-CoV-2 infection

Abstract

The human respiratory tract is lined with airway and alveolar epithelium. The respiratory epithelium, especially the airway epithelium, is the primary site of infection by respiratory viruses, including SARS-CoV-2. Productive infection of SARS-CoV-2 in the respiratory epithelial cells constitutes the basis of viral transmission and pathogenesis in humans. Yet a robust and well-characterized in vitro model of the respiratory epithelium remains elusive. We previously established long-term expanding human lung organoids from lung tissues and developed a proximal differentiation protocol to generate mucociliary airway organoids. However, a robust organoid model of the alveolar epithelium remains elusive.

Here we defined a distal differentiation protocol to generate alveolar organoids from the same source for generating airway organoids. The alveolar organoids, consisting of alveolar type I and type II epithelial cells, morphologically and functionally recapitulate the alveolar epithelium. We demonstrated that the immature “AT2” cells maintained in the expanding lung organoids served as the progenitor cell and gave rise to mature alveolar organoids. The alveolar organoids sustained productive SARS-CoV-2 infection, yet a lower replicative fitness was observed compared to that in airway organoids. We further optimized the two-dimensional (2D) airway organoids by mimicking the physiological milieu of the airway epithelium. The optimized 2D airway organoids sustained enhanced SARS-CoV-2 replication, providing a robust and biologically active organoid model for recapitulating the high infectivity of SARS-CoV-2. Thus, we established a bi-potential organoid culture system that can reproducibly and robustly expand the airway and alveolar epithelium in vitro for modeling respiratory biology and pathology.

The latest single-cell RNA sequencing studies of human airways significantly advanced the understanding of human cells based on traditional approaches and demonstrated that the airway epithelium in the nasal cavity varied from that in the tracheobronchial region. Meanwhile, the nasopharyngeal epithelium, also known as the upper respiratory epithelium, is the entry portal and primary site for SARS-CoV-2 replication and transmission. We proceeded to establish and characterize a robust organoid culture system of the human nasal epithelium from easily accessible nasal epithelial cells to model SARS-CoV-2 upper respiratory infection. The derived nasal organoids were long-term expandable, albeit for a shorter duration than the lung organoids. We then defined protocols to generate 3D and 2D differentiated nasal organoids that faithfully simulate the nasal epithelium. The optimized 2D nasal organoids in slightly acidic pH represented the optimal and in vitro correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2 in the upper respiratory tract which is superior to the existing in vitro models.

Notably, the higher infectivity and replicative fitness of the Omicron variant than the ancestral strain were accurately recapitulated in these optimized 2D nasal and airway organoids. Moreover, SARS-CoV-2 induced ciliary damage and tight junction dissemblance in these 2D organoids. In conclusion, we established a bipotential lung organoid culture system and a nasal organoid culture system to reproducibly expand the entire human respiratory epithelium in vitro for modeling respiratory diseases, including COVID-19.