Serum-free Three-Dimensional In Vitro Alveolar Lung Model

Abstract

European regulations spotlight the 3Rs principle of “Reduction, Refinement and Replacement” of animal experiments and therefore promote in vitro models. One major component of cell culture medium is the Fetal Bovine Serum (FBS), which is collected from the blood of bovine foetuses. Consequently, serious ethical concerns regarding the welfare of the foetuses are raised. Besides these ethical issues, the use of animal serum in cell culture has other disadvantages, such as lot-to-lot variability, rising cost and potential source of microbial contaminants. The purpose of this thesis is to adapt cell lines being part of a three-dimensional in vitro alveolar lung model to serum-free medium. The model has been developed for sensitization studies and is composed of epithelial cells (A549), endothelial cells (EA.hy926), dendritic cells (THP-1) and macrophages (PMA-differentiated THP-1). The first three cell lines were sequentially adapted to medium containing decreasing amount of serum. THP-1 cells were completely adapted to serum-free medium while A549 and EA.hy926 cells were adapted to medium containing less than one percent of serum. The serum has been replaced by another supplement medium called XerumFree (XF). Different characterizations regarding cell morphology, cell growth, cell viability and cell functionality have been performed on these adapted cells. The results were always compared to control cells, which were cultured in medium supplemented with 10% of FBS. The cell growth is much slower for THP-1 cells and even flat for both adherent cells (A549 and EA.hy926). The cell morphology of THP-1 and EA.hy926 does not change while the morphology of A549 changes. Some cells detach from the surface forming floating cell aggregates and the remaining adherent cells seem more tightly packed. For the same concentration of toxicant, the cell viability is much smaller when the proportion of serum-supplement media is smaller than 5% for both adherent cells. Different cell functionality tests have been performed on THP-1 and A549 cells, as dendritic and epithelial cells, respectively. These tests do not show any difference with the control cells.