A Fast Scoring of Human Primary Respiratory Epithelia Grown at Air-Liquid Interface (ALI) to Assess Epithelial Morphology in Research and Personalized Medicine Settings

Abstract

Background: In recent years, increasingly complex ALI protocols involving specialized, albeit laboratory-specific media have been established, while at the same time, many studies compile the data of only a few ALI donors in spite of site-, protocol- and donor-specific differentiation.

Methods: We describe a simple morphology scoring protocol using histology material derived from epithelia grown on ALI inserts in parallel to other, more complex readouts.

Results: Among more than 100 ALI inserts derived from different donors, significant differences in layer score (p = 0.001) and goblet cell score (p = 0.002) were observed when ALI epithelia derived from explanted lung material were compared to trachea-derived ALI cultures. Cortisol withdrawal for the final 2 days of ALI cultures influenced goblet cell density (p = 0.001).

Conclusions: While the histology score provides less resolution than FACS- or OMICs- based single cell analyses, the use of a subportion of the ALI epithelia grown on inserts makes it feasible to combine morphology assessment and other readouts of the same insert. This allows us to control for basic ALI morphology in research and personalized medicine settings in order to assess and, if desired, control for the impact of ALI culture protocols, site- and donor-specific influences on outcome of studies of ALI-derived epithelia.

Keywords: air–liquid interface culture; goblet cells; morphology score; primary respiratory epithelia.

Figure 1

Representative examples for ALI layer score. These examples were selected from independent donor sources, except for examples 1 and 3 in layer score 1, which were derived from the same donor. Example 2 for a layer score of 2 was the only tissue which is fixed using methacarn, and all others were treated using formamide. Using a visual analogue scale (Supplementary Figure S1) as a reference, a layer score of 0 was assigned to 1–2 rows of cells, a layer score of 1 for 3–5 rows of cells, a layer score of 2 for 5–7 rows of cells and a layer score of 3 for thick epithelia with 7 or more rows of cells. The scalebar is 50 µm for all inserts.

Figure 2

Representative examples for ALI apoptosis score. These examples were selected from independent donor sources, except for example 2 and 3 in layer score 0, which were derived from the same tissue. Arrows show apoptosis which can be recognized by their disintegrating or fragmented cell nuclei in the initial stage or by the bright eosinophilic discoloration of the underlying cell in the later stages. Using a visual analogue scale (Supplementary Figure S1) as a reference, an apoptosis score of 0 was assigned to ALI epithelia with no or only one solitary apoptosis. For an apoptosis score of 1, more than one apoptosis, but few in total, were observed. An apoptosis score of 2 indicated increased apoptoses, some of which were grouped together. An apoptosis score of 3 indicated many observable apoptoses that were conspicuously arranged in larger groups. The scalebar is 50 µm for all inserts.

Figure 3

Representative examples for ALI goblet cell score. These examples were selected from independent donor sources. Arrows denote isolated goblet cells, which belong to the intraepithelial glandular cells and serve to protect the mucous membranes by producing mucus and secreting it apically. Finally, a mucin film is formed on the surface, which is continuously transported further via the ciliated epithelium (kinocilia). A frequently observed phenomenon is the loss of the kinocilia due to the apically directed goblet cells. Using a visual analogue scale (Supplementary Figure S1) as a reference, a goblet cell score of 0 was assigned to epithelia without goblet cells, a goblet score of 1 denotes a few goblet cells, whereas a goblet cell score of 2 was assigned to epithelia with a goblet cell density on the surface that was close to 50 percent. A goblet cell score of 3 was used to indicate well over 50 percent of goblet cells of the epithelial surface. The scalebar is 50 µm for all inserts.

Figure 4

Representative examples for ALI air inclusion score. These examples were selected from independent donor sources. Arrows denote air inclusions in the cell layer. Using a visual analogue scale (Supplementary Figure S1) as a reference, an air inclusion score of 0 was assigned to ALI epithelia without air inclusions, an air inclusion score of 1 indicated that only one single void was observed in the in the entire preparation, an air inclusion score of 2 was attributed to ALI epithelia with few voids, while an air inclusion score of 3 was attributed to ALI epithelia with several air inclusions in the examined tissue. The scalebar is 50 µm for all inserts.