Tests on the modelThe liquid growth medium with added e-cig vapor will come in contact with the cellular model, actually with 3D co-culture of alveolar epithelial cells (NCI-H441) and endothelial cells of the human pulmonary microcirculation (HPMEC).
Referential protocols defined by the international scientific literature will characterize duration and methodologies used in the exposure of the cellular model to the growth media, in order to produce results that will be completely comparable with the already available ones. The effects on cells will be measured at the end of the exposure time through three different investigation methods: cellular viability/toxicity test, trans-epithelial electrical resistance measurement and quantification of inflammatory mediators.
A) CELLULAR VIABILITY/TOXICITY TEST
The cellular viability will be analyzed using alamarBlue®, a proven and reliable cell viability indicator. The active ingredient of alamarBlue® is a nontoxic, cell permeable compound that is blue in color (resazurin). Normal reduction reactions of metabolically active cells convert resazurin into a bright pink (resorufin).
So upon entering cells, reazurin is reduced to resorufin only in viable cells, thereby generating a quantitative measure of viability. The amount of fluorescence produced is proportional to the number of living cells and corresponds to the cells metabolic activity. Damaged and nonviable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells. After incubation with alamarBlue®, the cellular model will be read on a spectrophotometer.
B) TRANSEPITHELIAL ELECTRICAL RESISTENCE (TEER) MEASUREMENTS
Tissues are created by cells stick together to create a firm adhesion matrix that is essential in maintaining the cellular structure and that enables messages transmission among cells.
The reduction of cell adhesion could be one of the toxic effects some substances can cause, with serious consequences on the tissue.
Cell adhesion can be measured as the capacity of cells to prevent the passage of electricity. Transepithelial Electrical Resistence (TEER) measurement is the most reliable method for evaluating and monitoring the tissue cultures and it consists of two electrodes applied over the cells structure: the higher the detected passage of electricity is, the greater will be the toxic effect of the compound that has been in contact with cells through the growth medium.
C) QUANTIFICATION OF INFLAMMATORY MEDIATORS
When contaminated by some stress factors (e.g. cigarette smoke, alcohol, viruses, pathogens, parasites, etc.), cells react releasing some molecules (the so-called inflammatory mediators) that provoke an inflammation. Among the most important mediators, those belonging to the family of interleukin (in particular IL-6 and IL-8) are to be reported because they are used in the evaluation of the inflammatory effect connected with the use of e-cigarette.
Inflammation is a very complex process, that is controlled by several factors and that can also involve organs. The purpose of inflammation is to eliminate the initial cause of cell injury, activating the immune system and the more efficient cells in the damaged tissue to solve the problem. This process takes some time but then it is normally closely regulated by the body and succeeds into complete healing (for example, just think at a wound or at a local infection).
Prolonged stress factor leads to a chronic inflammation that could simultaneous destroy and heal tissues as a continuous biological response to the harmful stimuli. This means that stress factors not only have direct injurious effects but also some collateral consequences due to the incessant stimulation of the immune system that can hit active cells of the body.
Numerous studies have already demonstrated that traditional cigarettes have remarkable effects from this point of view because of pro-inflammatory molecules contained in smoke and also because of the daily routine of smoking.
To evaluate whether e-liquids can cause the same effects as those of smoking on lung tissues, the level of two of interleukin 8 will be quantified using specific test based on Enzyme-Linked Immune Sorbent Assay (ELISA) both in apical compartment (where lung cells develop), and in basal compartment (where endothelial cells develop).