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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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TEER measurements and tight junction detection in P0 and P1 EBEC cultures at ALI. [a-b] Time-course of TEER development in P0 (a) and P1 (b) insert cultures; the abscissa indicates culture days before (negative) and after (positive) ALI creation (day 0); TEER values (Ω·cm2) at each time-point are expressed as mean ± SEM of n = 5 (P0) or n = 6 (P1); insets show TEER graphs of individual subjects. [c-d] Representative images of time-dependent immunofluorescence staining of P0 (c) and P1 (d) insert cultures for the tight junction protein ZO-1 (one black and two white squares: ALI day 0, immediately before ALI creation; two black and one white squares: intermediate time-point of the ALI-culture phase leading to bioelectrical stabilization; all black squares: mature and electrically stable ALI-cultures) (objective magnification: ×40). Nuclei are blue stained with DAPI.
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Figure 4: TEER measurements and tight junction detection in P0 and P1 EBEC cultures at ALI. [a-b] Time-course of TEER development in P0 (a) and P1 (b) insert cultures; the abscissa indicates culture days before (negative) and after (positive) ALI creation (day 0); TEER values (Ω·cm2) at each time-point are expressed as mean ± SEM of n = 5 (P0) or n = 6 (P1); insets show TEER graphs of individual subjects. [c-d] Representative images of time-dependent immunofluorescence staining of P0 (c) and P1 (d) insert cultures for the tight junction protein ZO-1 (one black and two white squares: ALI day 0, immediately before ALI creation; two black and one white squares: intermediate time-point of the ALI-culture phase leading to bioelectrical stabilization; all black squares: mature and electrically stable ALI-cultures) (objective magnification: ×40). Nuclei are blue stained with DAPI.

Mentions: P0 insert cultures were successfully established from all 6 horses, although marked inter-individual variations could be observed. The data from one horse differed largely from the others and, thus, was excluded. Under liquid-liquid-interface (LLI) condition, cells at P0 showed a high rate of proliferation and usually formed confluent layers within 3-5 d post-seeding. TEER increased rapidly and in a time-dependent manner and reached mean values of 490.87 Ω·cm2 (± 122.45; n = 5) at confluence (Figure 4a). When cultures were switched to ALI conditions (d 0), TEER decreased abruptly to 79.16 ± 6.82% (n = 5) between d 0 and 4. In three cultures, which had TEER values ranging between 247.18 and 352.91 Ω·cm2 (Figure 4a - inset), the switch to ALI induced pronounced decrease in TEER (90.08 ± 2.34% at d 1-3 post-ALI). The decrease in TEER values was accompanied with partial retraction of the cell layer forming holes and at later time points with retraction of the entire cell layer at the periphery. Nevertheless, recovery of these alterations was seen starting from d 4-7 of ALI, during which cell proliferation occurred and again TEER values increased. Complete layer confluence was again achieved between d 18-21 of ALI and TEER was stable with values of about 300 Ω·cm2. In the remaining two cultures, TEER values of 637.00 and 902.22 Ω·cm2 could be measured under LLI conditions (Figure 4a - inset) and switching to ALI resulted in a moderate but permanent TEER decrease (62.77 ± 0.82%, n = 2) without visible alteration in cell layer appearance. For these cultures, TEER values were stable at about 300 Ω·cm2 between day 4 and 9 of ALI. Mean TEER values of P0 cultures finally stabilized at 310.53 ± 18.62 (n = 5) for up to d 30 of ALI (Figure 4a); among them two cultures remained stable even for up to 60 d (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)

TEER measurements and tight junction detection in P0 and P1 EBEC cultures at ALI. [a-b] Time-course of TEER development in P0 (a) and P1 (b) insert cultures; the abscissa indicates culture days before (negative) and after (positive) ALI creation (day 0); TEER values (Ω·cm2) at each time-point are expressed as mean ± SEM of n = 5 (P0) or n = 6 (P1); insets show TEER graphs of individual subjects. [c-d] Representative images of time-dependent immunofluorescence staining of P0 (c) and P1 (d) insert cultures for the tight junction protein ZO-1 (one black and two white squares: ALI day 0, immediately before ALI creation; two black and one white squares: intermediate time-point of the ALI-culture phase leading to bioelectrical stabilization; all black squares: mature and electrically stable ALI-cultures) (objective magnification: ×40). Nuclei are blue stained with DAPI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: TEER measurements and tight junction detection in P0 and P1 EBEC cultures at ALI. [a-b] Time-course of TEER development in P0 (a) and P1 (b) insert cultures; the abscissa indicates culture days before (negative) and after (positive) ALI creation (day 0); TEER values (Ω·cm2) at each time-point are expressed as mean ± SEM of n = 5 (P0) or n = 6 (P1); insets show TEER graphs of individual subjects. [c-d] Representative images of time-dependent immunofluorescence staining of P0 (c) and P1 (d) insert cultures for the tight junction protein ZO-1 (one black and two white squares: ALI day 0, immediately before ALI creation; two black and one white squares: intermediate time-point of the ALI-culture phase leading to bioelectrical stabilization; all black squares: mature and electrically stable ALI-cultures) (objective magnification: ×40). Nuclei are blue stained with DAPI.
Mentions: P0 insert cultures were successfully established from all 6 horses, although marked inter-individual variations could be observed. The data from one horse differed largely from the others and, thus, was excluded. Under liquid-liquid-interface (LLI) condition, cells at P0 showed a high rate of proliferation and usually formed confluent layers within 3-5 d post-seeding. TEER increased rapidly and in a time-dependent manner and reached mean values of 490.87 Ω·cm2 (± 122.45; n = 5) at confluence (Figure 4a). When cultures were switched to ALI conditions (d 0), TEER decreased abruptly to 79.16 ± 6.82% (n = 5) between d 0 and 4. In three cultures, which had TEER values ranging between 247.18 and 352.91 Ω·cm2 (Figure 4a - inset), the switch to ALI induced pronounced decrease in TEER (90.08 ± 2.34% at d 1-3 post-ALI). The decrease in TEER values was accompanied with partial retraction of the cell layer forming holes and at later time points with retraction of the entire cell layer at the periphery. Nevertheless, recovery of these alterations was seen starting from d 4-7 of ALI, during which cell proliferation occurred and again TEER values increased. Complete layer confluence was again achieved between d 18-21 of ALI and TEER was stable with values of about 300 Ω·cm2. In the remaining two cultures, TEER values of 637.00 and 902.22 Ω·cm2 could be measured under LLI conditions (Figure 4a - inset) and switching to ALI resulted in a moderate but permanent TEER decrease (62.77 ± 0.82%, n = 2) without visible alteration in cell layer appearance. For these cultures, TEER values were stable at about 300 Ω·cm2 between day 4 and 9 of ALI. Mean TEER values of P0 cultures finally stabilized at 310.53 ± 18.62 (n = 5) for up to d 30 of ALI (Figure 4a); among them two cultures remained stable even for up to 60 d (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