Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Airway branching generations in human and rat investigated in this study included BG0-6 in humans and BG0-5 in rats. b Workflow for imaging luminal epithelial cell type composition and ciliary beat and clearance function in airway samples. Schematic: Created in BioRender. Nawroth, J. (2025) https://BioRender.com/a01i578 ). c Example IF staining of cilia (ATUB, magenta) and secretory cells (SCGB1A1, green; MUC5AC, gray) in human and rat airway epithelium in BG0 (trachea) and BG5/6. Scale bar: 20 µm. d Quantification of luminal cell proportions labeled with ATUB (ciliated cells) or with MUC5AC and/or SCGB1A1 (secretory cells) as a function of airway branching generation in human and rat airway epithelium. Inset: Percentage of human secretory cell population positive for only MUC5AC (gray), only for SCGB1A1 (green), or for both (white) as a function of branching generation. Solid line: mean, shaded region: SEM. Numbers of human donors (2–3 FOVs each): BG0, n = 3; BG1, n = 3; BG2, n = 5; BG3, n = 3; BG4, n = 7; BG5, n = 2; BG6, n = 2; Numbers of rat donors (2–3 FOVs each): BG0, n = 5; BG1, n = 3; BG2, n = 2; BG4, n = 1; BG5, n = 1. For full donor information see Supplementary Tables and . Source data for ( d ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques: Imaging, Staining, Labeling

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Representative measurement of ciliary beat frequency (CBF) and associated particle clearance trajectories and speed in a human airway epithelial sample (BG2). b Same measurements in rat airway epithelial sample (BG1). Scalebar in ( a , b ): 100 µm. c Quantification of average CBF, particle clearance speed, and clearance per beat (CPB) in human airways BG0-6 and rat airways BG0-1. Number of human donors: n = 4. Number of rat donors: n = 6. d left: Clearance directionality as a function of distance in human (red) and rat (blue) airways. Thick lines are average curves. Right: Mean directionality over a flow distance of 80 µm. Number of human donors n = 4; number of rat donors: n = 4. Boxplots: Each solid dot is the mean value of one donor (1–3 BGs, 2–4 FOVs each); red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum; significance was assessed with two-sided unpaired t-test. Source data for ( c , d ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques:

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Representative IF-images of primary human airway epithelial cultures from 1 donor grown in different differentiation media for 28 days at ALI and stained for cilia (ATUB, magenta) and secretory cell markers (SCGB1A1, green; MUC5AC, white). Scalebar, 40 µm. b Average luminal cell type composition based on IF-staining (in vitro: N = 4–7 donors, 2 inserts each with 3–8 FOVs each; ex vivo: 9 donors, 1–3 BGs, 2–3 FOVs each). c Mapping of IF-staining data of in vitro and ex vivo samples onto three dimensions. y- axis: percentage of MUC5AC+ cells; x-axis: cilia coverage, i.e., percentage of ciliated (ATUB + ) cells; circle diameter: ratio of SCGB1A1+ to MUC5AC+ cell percentages. d Average percentage of MUC5B expressing cells and e relative percentage of MUC5B expressing cells that also express either SCGB1A1 or MUC5AC from IF-stainings (in vitro: n = 2 (SAGM, BD) to n = 4 (PC, PC-S, mAir) donors, 1–2 inserts each with 1–6 FOVs each; ex vivo: n = 3 donors, BG0, 2–4 FOVs each). Boxplot: Each dot represents mean of one donor; red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum. Source data for ( b – e ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques: Staining, In Vitro, Ex Vivo, Expressing

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Quantitative analysis of particle CPB and directionality in primary human airway epithelial cultures grown in different differentiation media for 28 days at ALI compared to human and rat benchmark data. Number of donors (2–3 inserts each with 6 FOVs each) for BD, mAir, SAGM, PC, PC-S, respectively: n = 5, 4, 4, 5, 4. b Quantitative analysis of ciliary beat metrics in airway cells cultured and visualized as in ( a ). Number of donors (2–3 inserts each with 6 FOVs each) for BD, mAir, SAGM, PC, PC-S, respectively: Cilia Coverage, n = 7, 4, 4, 7, 4; Ciliary Beat OP, n = 4, 4, 2 (1 not measurable), 4, 4; Ciliary Beat Amplitude: n = 4, 4, 3, 4, 4; Cilia Length, n = 4, 4, 4, 4, 4; Ciliation Gap, n = 4, 4, 3, 4, 4; Crystalline OP, n = 4, 4, 3, 4, 4. Boxplots in ( a , b ): Each solid dot is the mean value of one donor (1–3 BGs, 2–4 FOVs each); red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum. Dotted lines indicate average human and rat benchmark values. c Top: Predicted CPB and clearance directionality in in vitro cultures compared to predicted human airway performance (red). Shaded regions indicate uncertainty based on spread of input metrics. Measured mean values per donor are overlaid (dots). Bottom: Predicted change in CPB and directionality of in vitro cultures in different media if an individual cilia input parameter is set to match ex vivo values. Source data for ( a – c ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques: Cell Culture, In Vitro, Ex Vivo

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Airway branching generations in human and rat investigated in this study included BG0-6 in humans and BG0-5 in rats. b Workflow for imaging luminal epithelial cell type composition and ciliary beat and clearance function in airway samples. Schematic: Created in BioRender. Nawroth, J. (2025) https://BioRender.com/a01i578 ). c Example IF staining of cilia (ATUB, magenta) and secretory cells (SCGB1A1, green; MUC5AC, gray) in human and rat airway epithelium in BG0 (trachea) and BG5/6. Scale bar: 20 µm. d Quantification of luminal cell proportions labeled with ATUB (ciliated cells) or with MUC5AC and/or SCGB1A1 (secretory cells) as a function of airway branching generation in human and rat airway epithelium. Inset: Percentage of human secretory cell population positive for only MUC5AC (gray), only for SCGB1A1 (green), or for both (white) as a function of branching generation. Solid line: mean, shaded region: SEM. Numbers of human donors (2–3 FOVs each): BG0, n = 3; BG1, n = 3; BG2, n = 5; BG3, n = 3; BG4, n = 7; BG5, n = 2; BG6, n = 2; Numbers of rat donors (2–3 FOVs each): BG0, n = 5; BG1, n = 3; BG2, n = 2; BG4, n = 1; BG5, n = 1. For full donor information see Supplementary Tables and . Source data for ( d ) are provided as a file.

Article Snippet: Cells were then seeded at a density of 30 K cells cm -2 on PureCol (Advanced Biomatrix #5005) coated tissue culture dishes in airway epithelial growth media (Promocell #C-21160) and passaged at 80% confluence.

Techniques: Imaging, Staining, Labeling

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Representative measurement of ciliary beat frequency (CBF) and associated particle clearance trajectories and speed in a human airway epithelial sample (BG2). b Same measurements in rat airway epithelial sample (BG1). Scalebar in ( a , b ): 100 µm. c Quantification of average CBF, particle clearance speed, and clearance per beat (CPB) in human airways BG0-6 and rat airways BG0-1. Number of human donors: n = 4. Number of rat donors: n = 6. d left: Clearance directionality as a function of distance in human (red) and rat (blue) airways. Thick lines are average curves. Right: Mean directionality over a flow distance of 80 µm. Number of human donors n = 4; number of rat donors: n = 4. Boxplots: Each solid dot is the mean value of one donor (1–3 BGs, 2–4 FOVs each); red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum; significance was assessed with two-sided unpaired t-test. Source data for ( c , d ) are provided as a file.

Article Snippet: Cells were then seeded at a density of 30 K cells cm -2 on PureCol (Advanced Biomatrix #5005) coated tissue culture dishes in airway epithelial growth media (Promocell #C-21160) and passaged at 80% confluence.

Techniques:

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Representative IF-images of primary human airway epithelial cultures from 1 donor grown in different differentiation media for 28 days at ALI and stained for cilia (ATUB, magenta) and secretory cell markers (SCGB1A1, green; MUC5AC, white). Scalebar, 40 µm. b Average luminal cell type composition based on IF-staining (in vitro: N = 4–7 donors, 2 inserts each with 3–8 FOVs each; ex vivo: 9 donors, 1–3 BGs, 2–3 FOVs each). c Mapping of IF-staining data of in vitro and ex vivo samples onto three dimensions. y- axis: percentage of MUC5AC+ cells; x-axis: cilia coverage, i.e., percentage of ciliated (ATUB + ) cells; circle diameter: ratio of SCGB1A1+ to MUC5AC+ cell percentages. d Average percentage of MUC5B expressing cells and e relative percentage of MUC5B expressing cells that also express either SCGB1A1 or MUC5AC from IF-stainings (in vitro: n = 2 (SAGM, BD) to n = 4 (PC, PC-S, mAir) donors, 1–2 inserts each with 1–6 FOVs each; ex vivo: n = 3 donors, BG0, 2–4 FOVs each). Boxplot: Each dot represents mean of one donor; red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum. Source data for ( b – e ) are provided as a file.

Article Snippet: Cells were then seeded at a density of 30 K cells cm -2 on PureCol (Advanced Biomatrix #5005) coated tissue culture dishes in airway epithelial growth media (Promocell #C-21160) and passaged at 80% confluence.

Techniques: Staining, In Vitro, Ex Vivo, Expressing

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Quantitative analysis of particle CPB and directionality in primary human airway epithelial cultures grown in different differentiation media for 28 days at ALI compared to human and rat benchmark data. Number of donors (2–3 inserts each with 6 FOVs each) for BD, mAir, SAGM, PC, PC-S, respectively: n = 5, 4, 4, 5, 4. b Quantitative analysis of ciliary beat metrics in airway cells cultured and visualized as in ( a ). Number of donors (2–3 inserts each with 6 FOVs each) for BD, mAir, SAGM, PC, PC-S, respectively: Cilia Coverage, n = 7, 4, 4, 7, 4; Ciliary Beat OP, n = 4, 4, 2 (1 not measurable), 4, 4; Ciliary Beat Amplitude: n = 4, 4, 3, 4, 4; Cilia Length, n = 4, 4, 4, 4, 4; Ciliation Gap, n = 4, 4, 3, 4, 4; Crystalline OP, n = 4, 4, 3, 4, 4. Boxplots in ( a , b ): Each solid dot is the mean value of one donor (1–3 BGs, 2–4 FOVs each); red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum. Dotted lines indicate average human and rat benchmark values. c Top: Predicted CPB and clearance directionality in in vitro cultures compared to predicted human airway performance (red). Shaded regions indicate uncertainty based on spread of input metrics. Measured mean values per donor are overlaid (dots). Bottom: Predicted change in CPB and directionality of in vitro cultures in different media if an individual cilia input parameter is set to match ex vivo values. Source data for ( a – c ) are provided as a file.

Article Snippet: Cells were then seeded at a density of 30 K cells cm -2 on PureCol (Advanced Biomatrix #5005) coated tissue culture dishes in airway epithelial growth media (Promocell #C-21160) and passaged at 80% confluence.

Techniques: Cell Culture, In Vitro, Ex Vivo