In vitro modelling of bacterial pneumonia: a comparative analysis of widely applied complex cell culture models

Abstract

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host-pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air-liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models.

Keywords: advanced cell culture models; air–liquid-interface; bacterial pneumonia; lung-on-a-chip; organoid; spheroid.

Figure 1.

Overview of the most commonly used in vitro cellular models in pneumonia research. (A) While still broadly used, the standard two-dimensional submerged model has a lower translational value compared to the more advanced cellular models. (B) ALI models allow for cell differentiation by exposing cell monolayers to air resulting in a pseudostratified epithelium. (C) Spheroid models have a three-dimensional structure but lack self-renewing capacity. (D) Organoids are more complex three-dimensional cell structures with an organ-like architecture, including tissue lumen and self-renewal. (E) LOC devices contain multiple chambers where cells are seeded and exposed to a dynamic microenvironment, including a continuous air and medium flow, and fluid shear stress.

Figure 2.

Overview of the 84 selected research articles making use of a complex cellular infection model for bacterial pneumonia as of July 2023. (A) Articles subdivided per bacterium and cell model. (B) Selected articles per publication year, subdivided per advanced cellular model. ALI = air–liquid interface, LOC = lung-on-a-chip.

Figure 3.

Scanning electron microscopy (SEM) of human, upper respiratory epithelium. Transwell inserts with cells were bought from Epithelix (MucilAir) and cultured at the air–liquid-interface for 2 weeks before imaging. Homogeneous monolayer of bronchial epithelial cells, at different magnifications. (A) SEM at 600x magnification. Cilia are densely packed on the surface, (B) SEM at 2000x magnification. Cilia are densely packed on the surface, (C) SEM at 2000x magnification. Densely packed cilia (left arrow) interspersed with goblet cells (right arrow), and (D) SEM at 5000x magnification. Detailed close-up view of ciliated cells. Images were taken at the Advanced Centre for Advanced Microscopy facility of the University of Antwerp in collaboration with the Laboratory for Microbiology, Parasitology and Hygiene.

Figure 4.

(A) Main cells of the differentiated bronchial and alveolar epithelium. The respiratory epithelium is characterized by a multitude of cell subsets, including ciliated cells and serous goblet cells, club cells and basal progenitor cells. More rare cell types include the ionocytes, microfold cells, neuroendocrine cells, and tuft cells. The alveolar epithelium mainly consists of two cell types, the alveolar type I (ATI) and type II cells (ATII). (B) Cell lines used to establish complex bacterial infection models and some key characteristics are depicted. CFTR = cystic fibrosis transmembrane conductance regulator.

Figure 5.

Concise overview of nonendpoint assays. When developing an advanced infection cell model, implementing nonendpoint read-out experiments can help to obtain reliable, dynamic information of a singular cell infection condition throughout the duration of the infection, avoiding the use of multiple dependent replicates. Nonendpoint readouts can focus on both cellular (left-hand side) or bacterial (right-hand side) visualization, quantification, and activity.