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Development of a co-culture system for studies of airway epithelial and dendritic cell interactions

Nynäs, Anna-Lovisa LU (2015) KIM820 20151
Educational programmes
Department of Immunotechnology
Abstract
The interactions between airway epithelial cells (AECs) and dendritic cells (DCs) play an important role in the sensitization towards airborne allergens. Not much is known about the underlying mechanisms and novel in vitro approaches are needed, which include the different involved cell types in order to investigate what happens at the molecular level during sensitization. In this project, one aim was to develop a co-culture system with the bronchial epithelial cell line 16HBE14o- and monocyte-derived dendritic cells (MoDCs). Further, the suitability of using immunofluorescence (IF) and transepithelial electrical resistance (TEER) for controlling the phenotype of 16HBE14o- was investigated. Another aim was to investigate allergen-induced... (More)
The interactions between airway epithelial cells (AECs) and dendritic cells (DCs) play an important role in the sensitization towards airborne allergens. Not much is known about the underlying mechanisms and novel in vitro approaches are needed, which include the different involved cell types in order to investigate what happens at the molecular level during sensitization. In this project, one aim was to develop a co-culture system with the bronchial epithelial cell line 16HBE14o- and monocyte-derived dendritic cells (MoDCs). Further, the suitability of using immunofluorescence (IF) and transepithelial electrical resistance (TEER) for controlling the phenotype of 16HBE14o- was investigated. Another aim was to investigate allergen-induced changes in cytokine expression patterns in supernatants of 16HBE14o- cells, the primary AEC model MucilAir™, MoDCs and the dendritic cell line MUTZ-3 in order to compare these cell models.
MoDCs and 16HBE14o- were co-cultured in different media for the purpose of determining which cell media was optimal for both cell types in relation to growth and preservation of appropriate cellular phenotypes. The expression of activation markers on MoDCs was analyzed using flow cytometry, and 16HBE14o- were monitored using light microscopy, IF and TEER. Furthermore, 16HBE14o- and MoDCs were separately stimulated with protease allergens. The MoDC phenotype was analyzed by flow cytometry and the supernatants were collected. These, and the supernatants of MucilAir™ and MUTZ-3, stimulated with the allergens by others, were analyzed with regard to their cytokine content using a Human Cytokine 30-plex panel in a BioPlex200 instrument.
Routines of controlling the phenotype of 16HBE14o- cells using IF and TEER were established during this project. A RPMI-based media, called “RPMI-mod 3”, was concluded to be the best option for co-cultures with 16HBE14o- and MoDCs and the system was also optimized with regard to cell seeding densities, both in traditional cell culture wells and transwell inserts. Interestingly, co-culturing of MoDCs with 16HBE14o- in transwell inserts resulted in activation of MoDC. A first screen of cytokine regulation in response to different allergens in the four cell systems was performed and revealed clear differences between primary cells versus cell lines and AEC versus DC systems. This will allow further more focused follow-up studies in order to increase the understanding of the molecular interactions between AECs and DCs and in addition, it may pave the way for the development of a co-culture based in vitro prediction model. (Less)
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author
Nynäs, Anna-Lovisa LU
supervisor
organization
course
KIM820 20151
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Co-culture of epithelial and dendritic cells, Immunofluorescence, TEER, Monocyte-derived dendritic cell, Sensitization, 16HBE14o-, Epithelial cell, Allergy, Dendritic cells
language
English
id
8301783
date added to LUP
2015-12-07 14:28:26
date last changed
2015-12-07 14:28:26
@misc{8301783,
  abstract     = {The interactions between airway epithelial cells (AECs) and dendritic cells (DCs) play an important role in the sensitization towards airborne allergens. Not much is known about the underlying mechanisms and novel in vitro approaches are needed, which include the different involved cell types in order to investigate what happens at the molecular level during sensitization. In this project, one aim was to develop a co-culture system with the bronchial epithelial cell line 16HBE14o- and monocyte-derived dendritic cells (MoDCs). Further, the suitability of using immunofluorescence (IF) and transepithelial electrical resistance (TEER) for controlling the phenotype of 16HBE14o- was investigated. Another aim was to investigate allergen-induced changes in cytokine expression patterns in supernatants of 16HBE14o- cells, the primary AEC model MucilAir™, MoDCs and the dendritic cell line MUTZ-3 in order to compare these cell models.
MoDCs and 16HBE14o- were co-cultured in different media for the purpose of determining which cell media was optimal for both cell types in relation to growth and preservation of appropriate cellular phenotypes. The expression of activation markers on MoDCs was analyzed using flow cytometry, and 16HBE14o- were monitored using light microscopy, IF and TEER. Furthermore, 16HBE14o- and MoDCs were separately stimulated with protease allergens. The MoDC phenotype was analyzed by flow cytometry and the supernatants were collected. These, and the supernatants of MucilAir™ and MUTZ-3, stimulated with the allergens by others, were analyzed with regard to their cytokine content using a Human Cytokine 30-plex panel in a BioPlex200 instrument.
Routines of controlling the phenotype of 16HBE14o- cells using IF and TEER were established during this project. A RPMI-based media, called “RPMI-mod 3”, was concluded to be the best option for co-cultures with 16HBE14o- and MoDCs and the system was also optimized with regard to cell seeding densities, both in traditional cell culture wells and transwell inserts. Interestingly, co-culturing of MoDCs with 16HBE14o- in transwell inserts resulted in activation of MoDC. A first screen of cytokine regulation in response to different allergens in the four cell systems was performed and revealed clear differences between primary cells versus cell lines and AEC versus DC systems. This will allow further more focused follow-up studies in order to increase the understanding of the molecular interactions between AECs and DCs and in addition, it may pave the way for the development of a co-culture based in vitro prediction model.},
  author       = {Nynäs, Anna-Lovisa},
  keyword      = {Co-culture of epithelial and dendritic cells,Immunofluorescence,TEER,Monocyte-derived dendritic cell,Sensitization,16HBE14o-,Epithelial cell,Allergy,Dendritic cells},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of a co-culture system for studies of airway epithelial and dendritic cell interactions},
  year         = {2015},
}