Please use this identifier to cite or link to this item: http://hdl.handle.net/1946/15800
The respiratory system epithelium, including the bronchi and lungs, consists of several different types of epithelial cells. The upper respiratory tract has a pseudostratified epithelium and the epithelium grows thinner in the more distal respiratory tract ending in a simple epithelium in the alveoli where the gas exchange occurs. The epithelium of the upper respiratory tract is composed of various types of epithelial cells such as ciliated, goblet and basal cells. The basal cells are thought to be stem cells in the airways and the more differentiated cells of the epithelium arise from them. The basal cells serve as a reserve cell population that divides asymmetrically in response to injury. Research on the attributes of epithelial cells in vitro is important as the results can give information on how the cells of the bronchi and lungs develop. In these studies it is beneficial to use both primary cells and established cell lines.
Primary cells are cultured from donor tissue and are thought to represent the cell of origin better than established cell lines that have been genetically altered. Established cell lines have been grown for a long time in artificial environments, both in cell culture media and in two dimensional culture. Primary cells have been a part of a living being and reflect in part the state of the body they are taken from. However, they have a limited lifespan, as they have not been immortalized like the established cell lines and reproducing results can be difficult because of variations between donors.
In this study, primary epithelial cells were cultured from 19 bronchial tissue samples. The marker expression showed considerable donor variation and indicated a change in marker expression by time and passage. There seems to be a tendency for enrichment of p63 positive cells after first passage and mucin expression that identifies goblet cells does not seem to be present in passages after the initial one.
The VA10 cell line is a bronchial epithelial cell line that has a marker expression corresponding to basal cells. The VA10 cells are p63 positive, a basal cell marker, generating branching bronchioalveolar-like structures and pseudostratified epithelium in 3D culture and air-liquid culture, respectively. The VA10 cells are a heterogeneous group with a broad surface marker expression and a part of the project was to test if the cell line could be enriched for more basal cell properties by using a cell sorting technique based on antibodies against surface proteins expressed on basal cells. Subgroups of cells that had a high expression of basal cell markers NGFR and β4 integrin respectively were isolated. They showed a reduction in growth and a marker expression profile similar to that of the parental VA10 cells.
The VA10 cells were immortalized with the E6 and E7 genes, which degrade and bind p53 and retinoblastoma protein respectively. The original VA10 cell line forms branching structures with small cavities but their lumen formation is flawed. The E6 and E7 genes were knocked down with a shRNA construct in an effort to make VA10 cells that would have an improved lumen formation in branching structures in Matrigel. The knockdown of E6 and E7 was successful but when the shRNA was activated, the branching formation in Matrigel was severely reduced.
This study describes the characterization of freshly isolated primary cells from the human bronchus. The results underline the importance of using primary cells from multiple donors because of donor variance. Additionally, the cells are quite dynamic when it comes to surface marker expression, potentially changing as time passes. It is important to try and further our understanding of human primary cells to make them more useful as research tools. At the same time it would be beneficial to understand the strengths and limitations of both primary cells and cells lines since the best experiments would undoubtedly be conducted by combining the strengths of both for the most accurate conclusions.
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