How complex should an in vitro model be? Evaluation of complex 3D alveolar model with transcriptomic data and computational biological network models_suppl

Diego Marescotti
2019 ALTEX: Alternatives to Animal Experimentation  
as possible but as complex as necessary for the intended application. Compared with two-dimensional (2D) cultures, 3D cell cultures allow cells to grow or interact with their surroundings in all three dimensions in an artificial environment. 3D cultures are in vitro cultures in which primary cells, immortalized cell lines, stem cells, or explants are (a) grown using 3D scaffolds of various compositions, (b) allowed to organize themselves into a 3D structure (e.g., organoids), or (c) seeded on
more » ... th sides of Transwell ® inserts. Compared with 2D cultures, cells cultured in 3D better exhibit basic biological functions, such as viability, morphology, proliferation, differentiation, response to stimuli, Introduction The past decades have seen a surge in the development of in vitro models driven by 3R efforts, and models available today range in complexity from single cell type monolayers to multi-cell type, three-dimensional (3D) models (Duval et al., 2017; Edmondson et al., 2014) . It is generally acknowledged that more complex systems better reflect the in vivo situation (Braakhuis et al., 2015) ; however, this general assumption should be verified on a case-by-case basis, and the biological contribution derived from the incorporation of each additional cell type should be evaluated carefully. Overall, in vitro cellular models should be as simple Abstract To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This allowed us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types, and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-andeffect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages, which decreased the basal inflammatory and cell stress status of the co-culture. We compared transcriptional profiles from publicly available datasets of other in vitro models representing the airways and of healthy human lung tissue with those of our model. We found an increasing correlation between airway models and normal human lung tissue as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International license (, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is appropriately cited.
doi:10.14573/altex.1811221s fatcat:es6qo6uapbaizlp46xxgeumbdi