1. Planteamiento del problema
3.3 Marco conceptual o referencial
Biopsies and other ex-vivo fixed tissue samples such as whole organ excisions are a valuable resource in the understanding of normal organ function, and the pathological changes which occur in disease. They provide snapshots of a certain time and location in the disease, and provided they are stored correctly can be analysed many years in the future. They also allow researchers to look at the tissue as a whole, rather than as isolated cell types, revealing changes in structural relationships between different cell layers and structures. Unlike cell culture, these samples do not have the same risk of changes over time; they are not
continuously growing and they are an accurate representation of the tissue at the time of collection. However, biopsies and other fixed samples can be negatively affected by the fixation process, which can produce artefacts, destroy or alter certain markers or types of molecule and, depending on the method chosen, render the tissue unsuitable for certain analyses. For example, the commonly used fixation solution paraformaldehyde forms strong cross-links between proteins in the cell which then require reversal using antigen retrieval [260], and even then some proteins are too altered or damaged to be stained. In addition to molecular level changes, structural changes are also a product of collection and fixation; for example, lumen may collapse following collection, and this makes studying structures such as airways and alveoli challenging. Finally, although they do not change over time in the same way that cells do, prolonged storage may degrade antigen epitopes and other biopsy features. Despite these challenges, biopsies provide key insights into disease, and can often provide a starting point for identifying interesting processes and molecular changes which can direct and be further elucidated by cell culture studies.
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
137 Compared to biopsies, cell culture is extremely versatile, but at the cost of sacrificing a certain amount of in vivo accuracy. As previously mentioned (section 3.1) cells undergo changes during prolonged culture, which can reduce their relevance to the disease in
question. They also are isolated from the intercellular milieu of the body, and therefore may not accurately represent the full spectrum of disease, nor is it possible to ascertain if there are any knock-on effects in other important functional areas from seemingly minor changes in protein expression without complicated co-cultures.
However, the continual growth and proliferation of cell culture is also a boon – because they are still alive, the cells can be treated with drugs, cytokines or other molecules or compounds of interest, such as cigarette smoke, and their reactions observed. It is possible to treat cells with chemical substances it would be extremely difficult to administer to, or even get ethical approval to use on, humans. Cigarette smoke, and its effect on non-smokers’ cells, is a prime example, and the effects of drugs can be tested without concern for potential systemic side- effects.
Together, biopsies and cell culture can complement each other well, with biopsies providingproviding information on the gross physiological disease state, and cell culture providing theoretically free-rein on manipulation of micro-conditions and detailed
investigation and manipulation of pathways. It is very important to keep both sides in mind when researching processes such as EMT, and the comparison of culture with biopsy can potentially reveal if the cells truly represent the condition naturally seen in the organ of interest. In this vein, the main focus of this chapter is the question of how well primary cells taken from the airway epithelium maintain their phenotype in culture, and if the
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
138 commercially available BEAS-2B cell line is truly an accurate representation of healthy primary bronchial epithelial cells with reagards to the process of EMT.
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
139
4.1.4.
Chapter aimsThe aims of this chapter were as follows:
•
To establish the baseline differences between the epithelium in biopsies taken from non-smokers, smokers with normal lung function and people with airflow limitation with regards to markers of epithelial-mesenchymal transition (EMT).•
To confirm that cultured cells taken from non-smokers, smokers with normal lung function and people with airflow limitation maintain their disease phenotype in culture by comparing their pattern of expression to the biopsies with regards to markers of epithelial-mesenchymal transition (EMT).•
To further compare cells taken from non-smokers, smokers with normal lung function and people with airflow limitationlimitation, looking at the expression of additional EMT markers, two signalling molecules and two extracellular matrix remodelling factors.•
To evaluate the suitability of the commercially available, immortalised BEAS-2B cell line as a model for healthy bronchial epithelial cells with regards to markers of EMT, two signalling molecules and two extracellular matrix remodelling factors.Hypothesis: Airflow limitation will be marked by increased EMT signals and increased expression of signalling molecules and extracellular matrix remodelling proteins compared to
bronchial epithelial cells from non-smokers. Bronchial epithelial, while cells from smokers with normal lung function will exhibit an intermediate level of EMT, although and all
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
140 cultured cells will exhibit a slightly decreased intensity of EMT signals following culture and
cryopreservation compared to the biopsies. BEAS-2B cells will express similar levels of EMT-related markers as primary bronchial epithelial cells isolated from non-smokers.
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
141