Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media

Abstract

The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model

“…16HBE like Calu-3 are both airway-derived established epithelial cell lines, whose cells at confluence become polar with apical microvilli, form tight junctional seals and manifest cAMP-regulated CFTR (chloride) channels which are major contributors to the cell layers’ short-circuit current [ 29 , 30 ]. The 16HBE cell layers studied here developed TER values as high as 1000 Ohms × cm 2 , similar to the values seen with the Calu-3 airway epithelial cell line model [ 31 , 32 ] and normal bronchial epithelia in primary culture [ 33 ]. These values—obtained by culturing the cell layers under liquid (culture medium)—were found to be as high or higher than values obtained by air–liquid interface culture (approximately 300 Ohms × cm 2 ) [ 31 ], although Mathia et al (2002) [ 32 ] did report TER of 1000 Ohms × cm 2 using air-interface culture.…”

“…In addition to morphological and electrophysiological polarity, presence of apical CFTR chloride channels and presence of TJs, there are a wide range of other similarities among 16HBE, Calu-3, and primary bronchial epithelial cells, encompassing both structural proteins and regulatory pathways and proteins [ 34–38 ]. We would also point out that 16HBE does possess mucus-secreting capacity in part via the airway-specific mucin protein, MUC5AC, a further similarity to bronchial epithelia [ 39 , 33 ]. While 16HBE does lack cilia expression, it interestingly expresses the TRPV4 calcium channel thought to regulate cilia beating [ 40 ].…”

Targeting novel LSD1-dependent ACE2 demethylation domains inhibits SARS-CoV-2 replication

“…To verify the phenotypes observed in Caco-2 cells, we next used primary human bronchial epithelial cells obtained from healthy donors to assess airway epithelial changes following SARS-CoV-2 infection. The cells were differentiated at air–liquid interfaces (ALIs) to generate muco-ciliated tissue containing specialized bronchial epithelial cell subtypes, including ciliated cells (α-Tub + ), goblet cells (Muc5ac + ), and basal cells (KRT5 + ) 42 , 43 . There was a significant increase in infectious virus over 48 h in HBECs (Supplementary Fig.…”

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication

“…We next assessed whether SARS-CoV-2 induces PTGS2 in a more physiologically relevant cell culture system. We cultured primary human bronchial epithelial cells (HBECs) for 28 days at an air-liquid interface, which supports pseudostratified mucociliated differentiation providing an in vitro model of airway epithelium (15). We infected HBECs with SARS-CoV-2 at the apical surface of the culture and then performed single-cell RNA sequencing at 1, 2, and 3 days post infection (dpi) (16).…”