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Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions.

Abraham G, Zizzadoro C, Kacza J, Ellenberger C, Abs V, Franke J, Schoon HA, Seeger J, Tesfaigzi Y, Ungemach FR - BMC Vet. Res. (2011)

Bottom Line: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum.However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI.Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 15, 04103 Leipzig, Germany. gabraham@rz.uni-leipzig.de

ABSTRACT

Background: Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions.

Results: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures.

Conclusions: This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.

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Related in: MedlinePlus

Effectiveness of EBEC isolation procedure and purity of fresh isolates. [a-b] Representative H&E staining of equine bronchial tissue before (a) and after (b) 2 h exposure to 0.25% trypsin-EDTA (objective magnification: ×10). Arrows indicate the intact epithelium before digestion and the complete removal of epithelium from the underlying connective tissue after trypsin digestion. [c-d] Representative immunocytochemical staining of cytocentrifuged fresh EBEC for the epithelial cell markers cytokeratins 5/6/18 (c) and the mesenchymal cell marker vimentin (d) (objective magnification: ×40). Green fluorescence (FITC) stain indicates positive signal. Nuclei are blue stained with DAPI.
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Figure 1: Effectiveness of EBEC isolation procedure and purity of fresh isolates. [a-b] Representative H&E staining of equine bronchial tissue before (a) and after (b) 2 h exposure to 0.25% trypsin-EDTA (objective magnification: ×10). Arrows indicate the intact epithelium before digestion and the complete removal of epithelium from the underlying connective tissue after trypsin digestion. [c-d] Representative immunocytochemical staining of cytocentrifuged fresh EBEC for the epithelial cell markers cytokeratins 5/6/18 (c) and the mesenchymal cell marker vimentin (d) (objective magnification: ×40). Green fluorescence (FITC) stain indicates positive signal. Nuclei are blue stained with DAPI.

Mentions: Successful dissociation of epithelial cells was confirmed by H&E staining after short-term (2 h) incubation of native bronchial tissues with 0.25% trypsin-EDTA solution (Figure 1a, b). A mean yield of 15.25 ± 1.90 × 106 viable cells was obtained from 500 mg minced tissue, with viability of > 95% as assessed by trypan blue exclusion test (n = 12). Freshly dissociated cells often consisted of single or clumped and rotating ciliated cells. 92.28 ± 0.88% of these cells were CK5/6/18-positive indicating epithelial origin while only 9.43 ± 1.13% (n = 12) VIM-positive suggesting non-epithelial cell origin (Figure 1c, d). 100 cells were counted in at least three randomly selected fields.


Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions.

Abraham G, Zizzadoro C, Kacza J, Ellenberger C, Abs V, Franke J, Schoon HA, Seeger J, Tesfaigzi Y, Ungemach FR - BMC Vet. Res. (2011)

Effectiveness of EBEC isolation procedure and purity of fresh isolates. [a-b] Representative H&E staining of equine bronchial tissue before (a) and after (b) 2 h exposure to 0.25% trypsin-EDTA (objective magnification: ×10). Arrows indicate the intact epithelium before digestion and the complete removal of epithelium from the underlying connective tissue after trypsin digestion. [c-d] Representative immunocytochemical staining of cytocentrifuged fresh EBEC for the epithelial cell markers cytokeratins 5/6/18 (c) and the mesenchymal cell marker vimentin (d) (objective magnification: ×40). Green fluorescence (FITC) stain indicates positive signal. Nuclei are blue stained with DAPI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117700&req=5

Figure 1: Effectiveness of EBEC isolation procedure and purity of fresh isolates. [a-b] Representative H&E staining of equine bronchial tissue before (a) and after (b) 2 h exposure to 0.25% trypsin-EDTA (objective magnification: ×10). Arrows indicate the intact epithelium before digestion and the complete removal of epithelium from the underlying connective tissue after trypsin digestion. [c-d] Representative immunocytochemical staining of cytocentrifuged fresh EBEC for the epithelial cell markers cytokeratins 5/6/18 (c) and the mesenchymal cell marker vimentin (d) (objective magnification: ×40). Green fluorescence (FITC) stain indicates positive signal. Nuclei are blue stained with DAPI.
Mentions: Successful dissociation of epithelial cells was confirmed by H&E staining after short-term (2 h) incubation of native bronchial tissues with 0.25% trypsin-EDTA solution (Figure 1a, b). A mean yield of 15.25 ± 1.90 × 106 viable cells was obtained from 500 mg minced tissue, with viability of > 95% as assessed by trypan blue exclusion test (n = 12). Freshly dissociated cells often consisted of single or clumped and rotating ciliated cells. 92.28 ± 0.88% of these cells were CK5/6/18-positive indicating epithelial origin while only 9.43 ± 1.13% (n = 12) VIM-positive suggesting non-epithelial cell origin (Figure 1c, d). 100 cells were counted in at least three randomly selected fields.

Bottom Line: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum.However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI.Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 15, 04103 Leipzig, Germany. gabraham@rz.uni-leipzig.de

ABSTRACT

Background: Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions.

Results: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures.

Conclusions: This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.

Show MeSH
Related in: MedlinePlus