Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Abstract

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens.Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc.In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Airway epithelial dual oxidase 1 mediates allergen-induced IL-33 secretion and activation of type 2 immune responses

“…Murine tracheal epithelial (MTE) cells were isolated from either wild-type C57BL/6 mice or Duox1 −/− mice (25), and cultured as described previously (26, 27). Primary human nasal epithelial (HNE) cells were isolated from healthy volunteers and subjects with allergic rhinitis and cultured as recently described (28). Atopy was confirmed by positive skin tests and elevated serum IgE (> 100 IU/ml), asthma was confirmed by positive response to bronchodilator (≥ 200 cc and 12 % improvement in FEV1 and/or FVC) or a positive methacholine challenge test (PC 20 < 8mg/ml), and all selected subjects had rhinitis with an SNQ score >1 (29).…”

Novel Analysis of Immune Cells from Nasal Microbiopsy Demonstrates Reliable, Reproducible Data for Immune Populations, and Superior Cytokine Detection Compared to Nasal Wash

“…An improved method of sampling the nasal mucosa would lead to a greater understanding of the cellular components of the immune response in the nasopharynx. Cell collection using nasal curettes has previously been used to collect epithelial cells for culture, as well as for gene expression analysis [13, 14]. Nasal brushes have also been used to collect samples to investigate epithelial cell phenotype [15].…”

Phenotypic Modification of Human Airway Epithelial Cells in Air–Liquid Interface Culture Induced by Exposure to the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

“…When these biopsies obtained from healthy non-smoking human subjects are transitioned to transwell supports and propagated under ALI conditions the resulting cultures recapitulate the normal airway epithelial phenotype [11]. In this study, we obtained nasal epithelial biopsies from healthy, non-smokers and following mitotic expansion and transitioning them to ALI conditions began incorporating 100 mg/ml NNK into the culture medium.…”

“…The fresh biopsies were established in an in vitro environment and ALI cultures generated by standard techniques previously reported [11]. At the time the cultures were transitioned to ALI conditions, supplementation of the culture medium with 100 mg/ ml NNK (Chemsyn Laboratories, Lenexa, KS) was begun.…”