Periodic Reporting for period 1 - SCREENED (A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner)
Reporting period: 2019-01-01 to 2020-06-30
Five decellularization protocols suitable for removal of all cell types from the adult male and female, rat thyroid gland have been developed without hampering the main structural and geometrical 3D organisation of the thyroid lobe matrix: each showing a preferential retention of specific matrix molecules (collagen types, structural proteins, growth factors, glycosaminoglycans). In addition, isolation, expansion, enrichment and characterisation of adult male rat, thyroid stem cells / progenitor (TSC/P) using a simple, reliable and reproducible methodology has been achieved, based on long-term, semi-starvation low-density monolayer subcultures.
We also focused on the optimisation of microfluidic bioprinting for the generation of the bioprinted thyroid model. We evaluated different hydrogel based bioink formulations to assess their printability and the parameters for an optimal bioprinting in terms of fibre dimension and deposition. We verified that bioprinted cells remain viable and metabolically active inside the construct. We also increased bioprinted constructs’ robustness by post crosslinking the construct with a CaCl2 base solution. Finally, we exploited a new core-shell bioprinting technique that allowed to produce hollow fibers containing thyroid and endothelial cells at the same time. As a further method to provide better control of cell spatial positioning for the 3D cellular assays to be developed in SCREENED, we also optimized a method to magnetize cells while maintaining a high viability.
The current lack of available proteomic and transcriptomic information on the thyroid drove us to initiate the first combined proteomic and transcriptomic analysis of ED effects on (non-cancerous) thyroid cells, using state of the art technologies to identify thousands of gene and protein thyroid signatures. Preliminary data on recent research on the in vivo effects of a mixture of polychlorinated biphenyls on the hypothalamic-pituitary-thyroid (H-P-T) axis of adult male rats have provided groundwork for eventual development of theoretical modelling of in vivo EDs action on the rat thyroid gland.
We are also producing a bioprinted 3D model able to mimic the thyroid architecture and functionality. The main models currently available for thyroid studies are based on 2D cell cultures or on the use of animal models. Either of these two solutions do not represent the human thyroid complexity. With bioprinting, we could produce a 3D model able to closely mimic the human thyroid and to offer different advantages over classical 2D models. At the same time, the bioprinted model could reduce the use of animal models for drug screening and discovery purposes, while offering a new platform for studying thyroid physiology and physiopathology. The potential addition of magnetized cells as a further method to control cell spatial deposition in 3D would offer the capacity to precisely locate thyrocytes and endothelial cells in the 3D cellular assays of SCREENED. This method would allow an exquisite control in organoids and decellularized extracellular matrix constructs, as well as a further level of spatial control in bioprinted constructs.
Since there is no existing state of the art for (normal) thyroid cell transcriptomics and proteomics, we will establish a baseline transcriptome and proteome of human thyroid cells and the impact of ED’s on them. By the end of SCREENED we expect to collect comparative and reliable information between the effects of a list of EDs in the in vitro 3D thyroid assays under experimental development and in vivo results on rodent models (male and female rats) used as control systems.