1.1. Anatomy and Cellular Characteristics of the Human Respiratory System
The respiratory system is a complex network that is formed by the upper (nasal cavity, pharynx and larynx), and the lower respiratory tract, which is further divided into the proximal (trachea, bronchi and bronchioles) and distal airway (respiratory bronchioles and alveoli) (). The entire human respiratory tract, which is lined with polarized airway epithelium (apical side facing air/lumen, basolateral side facing the internal milieu), is structurally diverse and fulfills multiple functions, depending on anatomical location. While the proximal conducting airways function as a gas transport system, alveoli of the distal airway facilitate air exchange and respiration 
Organization and cellular characteristics of the human respiratory airway. Adapted from “Respiratory Epithelium”, by BioRender.com (accessed on 22 April 2021) (2021). Retrieved from https://app.biorender.com/biorender-templates
(accessed on 21 April 2021).
Structurally, the pseudostratified tracheobronchial epithelium consists of specialized cells including columnar club, ciliated, goblet and basal cells, incorporated into a basement membrane, which facilitate mucus production, mucociliary clearance, mucus secretion, and serve as progenitors for columnar cells 
. Moving further down the respiratory tree, thinner cuboidal epithelium appears with an increased number of club cells. In contrast, goblet cells become increasingly less frequent until they can no longer be found in the alveoli. The distal alveolar region is lined with two types of epithelial cells (alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2)). Squamous AT1 cells provide a specialized surface for gas exchange, whereas cuboidal AT2 cells secrete pulmonary surfactant to prevent alveolar collapse during expiration 
In addition to gas exchange, the human airway epithelium has been recognized to function as the main entry point for several pathogens. Notably, human airway epithelial cells represent a primary target of coronaviruses, including the highly transmissible and pathogenic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 
. To defend itself from pathogens such as SARS-CoV-2, epithelial cells have adapted numerous strategies that include tightly packed epithelial cells forming tight junctions, mucosal epithelium trapping invading pathogens in the secreted mucus barrier and the expression of pattern-recognition receptors. Furthermore, within the so-called mucociliary escalator, invaded pathogens are cleared by coordinated cilia beating 
. Additionally, cells of the immune system (e.g., macrophages) are present to eliminate the intruding pathogen 
Given the complexity and diversity of the human respiratory system, it is of utmost importance to develop authentic in vitro and ex vivo culture models, which faithfully model basic human pathology and the complexity and diversity of human respiratory tissues to study emerging viral respiratory pathogens, such as SARS-CoV-2.
Respiratory coronaviruses (CoVs) were first discovered in the 1960s 
and are commonly found in many vertebrates and humans, where they are known to cause various types of disease. CoVs are a large group of viruses belonging to the family Coronaviridae
, which are further divided by phylogenetic clustering into 4 genera: Alphacoronavirus
(γCoV) and Deltacoronavirus
So far, four endemic coronaviruses have been identified circulating in the human population, including HCoV-229E (αCoV), HCoV-OC43 (βCoV), HCoV-NL63 (αCoV) and HCoV-HKU1 (βCoV) that are usually causing mild, self-limiting respiratory infections also known as the “common cold” in immunocompetent patients and occasionally respiratory tract infections in immunosuppressed individuals 
. In contrast, several βCoVs with increasing pathogenicity have emerged during the past two decades. The Severe Acute Respiratory Syndrome CoV (SARS-CoV) outbreak in the Guangdong Province of China in 2002–2003 lead to over 8400 confirmed cases with a mortality rate of about 11% 
. In addition, Middle East Respiratory Syndrome CoV (MERS-CoV) emerged on the Arabian Peninsula 10 years later, in 2012, causing a serious series of highly pathogenic respiratory tract infections, leading to over 2800 verified cases with a mortality rate of 35% 
Most recently, SARS-CoV-2, a betacoronavirus with 96.2% sequence similarity to the bat CoV RaTG13 and 79% sequence similarity to SARS-CoV, emerged, leading to a fast-spreading global pandemic 
. As of 12 March 2021, over 119 million confirmed cases and nearly 2.6 million deaths were reported globally by the World Health Organization 
, demonstrating its remarkably high transmission potential and mortality.
The clinical course of SARS-CoV-2 infection varies significantly from patient to patient in its severity and disease outcome. While some patients experience mild symptoms (such as fever, cough, shortness of breath, sore throat and muscle ache) or are asymptomatic, in others it causes acute respiratory distress syndrome (ARDS), severe sepsis and even death 
. Notably, in some individuals, severe disease progression has been linked to the so-called “cytokine storms”, initiated through rapid virus propagation and uncontrolled inflammation 
SARS-CoV-2 is an enveloped, single-stranded positive-sense RNA virus which encodes a 29.9 kb genome. Structurally, virion particles are assembled from four proteins: The spike (S), small envelope (E), membrane (M) and nucleocapsid (N) glycoproteins are involved in host cell receptor recognition, virion assembly and egress, shaping the virion and viral RNA encapsulation, respectively. In addition, 16 non-structural proteins and a variety of accessory proteins have been identified to be involved in RNA processing, RNA replication, and survival in the host cell 
The main route of transmission is believed to be through respiratory droplets that are released into the air through breathing, coughing, sneezing or talking, a high risk especially upon close contact 
. SARS-CoV-2 then enters the human respiratory system and enters susceptible cells by binding of the surface spike (S) glycoprotein to its receptor, human angiotensin-converting enzyme 2 (ACE2), expressed on membranes of epithelial cells in the upper and lower airways with very high affinity 
. Subsequent proteolytic priming of the viral spike protein through cellular proteases (e.g., TMPRSS2) facilitates fusion of the viral envelope with the cell membrane and release of the viral genome into the cells 
. Afterwards, the virus replicates and spreads within the airways and alveoli.
Within the past year, several vaccines targeting SARS-CoV-2 were developed with about 82 candidates in clinical trials and 182 candidates currently in preclinical development, as of 17 March 2021 
. Notably, the first vaccine authorization (mRNA vaccine BNT162B (Pfizer-BioNTech) that encodes for the full-length SARS-CoV-2 spike protein) was issued in December 2020 after successful completion of the third clinical phase 
, and was followed by approval of numerous other vaccines (e.g., AZD1222 
, mRNA-1273 
). There are no specific antiviral molecules approved for the treatment of SARS-CoV-2, up until now, with the exception of the drug remdesivir, which has an emergency use authorization, however, it is no longer recommended by the WHO 
. This highlights the urgency of identifying and developing novel drug candidates, for which authentic cell culture models are of utmost importance.
Furthermore, the fact that CoVs are classified as zoonotic pathogens that can easily be transmitted from animals to humans implicates the possibility of future outbreaks, which necessitates future extensive studies of coronaviruses.
Recent studies have successfully utilized animal models, including non-human primates, human ACE2 transgenic mice and golden Syrian hamsters, to study SARS-CoV-2 infection 
(an in-depth literature review on animal models used for SARS-CoV-2 infection and COVID-19 can be found here 
Ever since the beginning of the COVID-19 pandemic, numerous human-derived in vitro and ex vivo models have been proposed for the study of SARS-CoV-2 pathogenesis and drug development.