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Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for <t>MUC5AC.</t> g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.
Muc5ac, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for <t>MUC5AC.</t> g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.
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Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for <t>MUC5AC.</t> g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.
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Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for <t>MUC5AC.</t> g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.
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Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for MUC5AC. g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.

Journal: ERJ Open Research

Article Title: Ciliated epithelial cell differentiation at air–liquid interface and respiratory syncytial virus infection using animal-free media and substrates

doi: 10.1183/23120541.00028-2025

Figure Lengend Snippet: Barrier integrity and ciliated epithelial cell differentiation of human bronchial epithelial cells (BECs) at air–liquid interface (ALI). a,b) Epithelial integrity of BECs differentiated from one donor at ALI on membrane inserts coated with human placenta collagen IV for 4 weeks (n=8) compared with our non-animal-free (AF)model (n=10) that contains an additional Matrigel (mouse-derived) layer. Barrier integrity was measured by trans-epithelial electrical resistance (TEER) (Ω·cm −2 ) (a) and dextran permeability (μg·mL −1 ) (b). Error bars represent mean± sem of n=8–10 independent Transwells. *: p<0.05 (unpaired ANOVA with Benjamini–Hochberg correction for multiple comparisons). c) Histogram of ciliary beat frequency (CBF) (Hz) was quantified via high-speed video microscopy of 6400 regions of interest (ROIs) from five independent areas (one airway epithelial cell (AEC) donor). The red dotted line represents the mean CBF value of all areas. d,e) Representative brightfield image (d) and activity map (e) of one area (field of view) to show the position and distribution of motile ciliated cells. Scale bar, 50 μm. f) Example flow cytometry gating strategy to determine the population of basal, ciliated and goblet cells in inverted ALI cultures. Basal cells were defined as the proportion of CD49f/CD127 double-positive cells in the live cell population. Ciliated cells were then gated from the nonbasal cell population based on acetyl-α-tubulin + and CD66c − expression, whereas goblet cells were further separated from the nonciliated population based on positive expression for MUC5AC. g) Proportion (%) of basal (CD49f + CD271 + ), ciliated (acetyl-α-tubulin + CD66c − ) and goblet (MUC5AC + ) cells from one AEC donor (n=3 wells). Red dot and error bars represent mean± sem of n=3 wells.

Article Snippet: MUC5AC , Goblet cells , PE , Miltenyi Biotec (130-127-552) , No , 1:50.

Techniques: Cell Differentiation, Membrane, Derivative Assay, Permeability, Microscopy, Activity Assay, Flow Cytometry, Expressing