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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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Morphological appearance of P0 and P1 EBEC liquid-liquid interface cultures before ALI creation. [a-b] Light microscopy of epithelial cell layers generated by freshly isolated cells (P0 insert cultures) (a) and by cells recovered from sub-confluent P0 cultures previously grown on solid supports in serum-containing medium (P1 insert cultures) (b) (objective magnification: ×4). [c-d] Representative scanning electron micrographs of P0 (c) and P1 (d) EBEC insert cultures: in P0 cultures (c) few ciliated cells, small amount of mucus and short microvilli can be observed; in P1 cultures (d) cells show poorly defined boundaries and many short microvilli. Scale bars = 2 μm. [e] Representative H&E staining of sections of epithelial layers grown on membrane inserts (objective magnification: ×20), showing that in both P0 and P1 cultures epithelial cells form a flat monolayer. Asterisk indicates the porous membrane support.
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Figure 5: Morphological appearance of P0 and P1 EBEC liquid-liquid interface cultures before ALI creation. [a-b] Light microscopy of epithelial cell layers generated by freshly isolated cells (P0 insert cultures) (a) and by cells recovered from sub-confluent P0 cultures previously grown on solid supports in serum-containing medium (P1 insert cultures) (b) (objective magnification: ×4). [c-d] Representative scanning electron micrographs of P0 (c) and P1 (d) EBEC insert cultures: in P0 cultures (c) few ciliated cells, small amount of mucus and short microvilli can be observed; in P1 cultures (d) cells show poorly defined boundaries and many short microvilli. Scale bars = 2 μm. [e] Representative H&E staining of sections of epithelial layers grown on membrane inserts (objective magnification: ×20), showing that in both P0 and P1 cultures epithelial cells form a flat monolayer. Asterisk indicates the porous membrane support.

Mentions: As assessed by light and electron microscopy, or by H&E staining and immunocytochemistry, P0 and P1 cells on membrane inserts showed similarities and differences depending on the conditions and age of the ALI culture (Figure 5). Regardless of confluence level, cells under LLI condition (Figure 5a, b), were large, uniform, with short microvilli and poorly defined borders (especially in P1 cultures) (Figure 5c, d) and formed a monolayered, flattened and almost undifferentiated epithelium (Figure 5e). Indeed, some ciliated cells could be observed in P0 cultures at this stage.


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)

Morphological appearance of P0 and P1 EBEC liquid-liquid interface cultures before ALI creation. [a-b] Light microscopy of epithelial cell layers generated by freshly isolated cells (P0 insert cultures) (a) and by cells recovered from sub-confluent P0 cultures previously grown on solid supports in serum-containing medium (P1 insert cultures) (b) (objective magnification: ×4). [c-d] Representative scanning electron micrographs of P0 (c) and P1 (d) EBEC insert cultures: in P0 cultures (c) few ciliated cells, small amount of mucus and short microvilli can be observed; in P1 cultures (d) cells show poorly defined boundaries and many short microvilli. Scale bars = 2 μm. [e] Representative H&E staining of sections of epithelial layers grown on membrane inserts (objective magnification: ×20), showing that in both P0 and P1 cultures epithelial cells form a flat monolayer. Asterisk indicates the porous membrane support.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Morphological appearance of P0 and P1 EBEC liquid-liquid interface cultures before ALI creation. [a-b] Light microscopy of epithelial cell layers generated by freshly isolated cells (P0 insert cultures) (a) and by cells recovered from sub-confluent P0 cultures previously grown on solid supports in serum-containing medium (P1 insert cultures) (b) (objective magnification: ×4). [c-d] Representative scanning electron micrographs of P0 (c) and P1 (d) EBEC insert cultures: in P0 cultures (c) few ciliated cells, small amount of mucus and short microvilli can be observed; in P1 cultures (d) cells show poorly defined boundaries and many short microvilli. Scale bars = 2 μm. [e] Representative H&E staining of sections of epithelial layers grown on membrane inserts (objective magnification: ×20), showing that in both P0 and P1 cultures epithelial cells form a flat monolayer. Asterisk indicates the porous membrane support.
Mentions: As assessed by light and electron microscopy, or by H&E staining and immunocytochemistry, P0 and P1 cells on membrane inserts showed similarities and differences depending on the conditions and age of the ALI culture (Figure 5). Regardless of confluence level, cells under LLI condition (Figure 5a, b), were large, uniform, with short microvilli and poorly defined borders (especially in P1 cultures) (Figure 5c, d) and formed a monolayered, flattened and almost undifferentiated epithelium (Figure 5e). Indeed, some ciliated cells could be observed in P0 cultures at this stage.

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