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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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Features of mucociliary differentiation of EBEC cultured on solid supports in serum-free or serum-containing medium. [a] PAS-positive (magenta coloured) cells in 30 days old EBEC cultures on glass cover slips in serum-containing medium (objective magnification: ×10); inset shows less intense PAS-positive stain in EBEC grown in serum-free medium (objective magnification: ×40). [b] Ciliated cells detected by phase contrast microscopy in 30 days old EBEC cultures on tissue culture flasks and glass cover slips in serum-containing medium; arrows indicate cilia (objective magnification: ×40).
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Figure 3: Features of mucociliary differentiation of EBEC cultured on solid supports in serum-free or serum-containing medium. [a] PAS-positive (magenta coloured) cells in 30 days old EBEC cultures on glass cover slips in serum-containing medium (objective magnification: ×10); inset shows less intense PAS-positive stain in EBEC grown in serum-free medium (objective magnification: ×40). [b] Ciliated cells detected by phase contrast microscopy in 30 days old EBEC cultures on tissue culture flasks and glass cover slips in serum-containing medium; arrows indicate cilia (objective magnification: ×40).

Mentions: In addition to the observed formation of tight junctions, EBEC cultures in serum containing AECGM exhibited also more typical features of differentiation (Figure 3). Many PAS-positive cells (likely mucus-producing cells) (Figure 3a) as well as many ciliated cells were routinely detected (Figure 3b; video images can be supplied upon request), even 24 h after plating and during the whole culture period (~ 30 days). In contrast, in serum-free AECGM, EBECs completely lacked visible cilia and showed less intense PAS-positivity (Figure 3a - inset).


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)

Features of mucociliary differentiation of EBEC cultured on solid supports in serum-free or serum-containing medium. [a] PAS-positive (magenta coloured) cells in 30 days old EBEC cultures on glass cover slips in serum-containing medium (objective magnification: ×10); inset shows less intense PAS-positive stain in EBEC grown in serum-free medium (objective magnification: ×40). [b] Ciliated cells detected by phase contrast microscopy in 30 days old EBEC cultures on tissue culture flasks and glass cover slips in serum-containing medium; arrows indicate cilia (objective magnification: ×40).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Features of mucociliary differentiation of EBEC cultured on solid supports in serum-free or serum-containing medium. [a] PAS-positive (magenta coloured) cells in 30 days old EBEC cultures on glass cover slips in serum-containing medium (objective magnification: ×10); inset shows less intense PAS-positive stain in EBEC grown in serum-free medium (objective magnification: ×40). [b] Ciliated cells detected by phase contrast microscopy in 30 days old EBEC cultures on tissue culture flasks and glass cover slips in serum-containing medium; arrows indicate cilia (objective magnification: ×40).
Mentions: In addition to the observed formation of tight junctions, EBEC cultures in serum containing AECGM exhibited also more typical features of differentiation (Figure 3). Many PAS-positive cells (likely mucus-producing cells) (Figure 3a) as well as many ciliated cells were routinely detected (Figure 3b; video images can be supplied upon request), even 24 h after plating and during the whole culture period (~ 30 days). In contrast, in serum-free AECGM, EBECs completely lacked visible cilia and showed less intense PAS-positivity (Figure 3a - inset).

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