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A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner

Periodic Reporting for period 2 - SCREENED (A multistage model of thyroid gland function for screening endocrine-disrupting chemicals in a biologically sex-specific manner)

Berichtszeitraum: 2020-07-01 bis 2021-12-31

Endocrine disrupting Chemicals (EDCs) are commonly found in our everyday life, but there is growing evidence that EDCs interfere with the functioning of the thyroid and cause changes in thyroid hormone concentrations, the peripheral metabolism of these hormones and the signalling of their receptors. The mechanism by which they act on the thyroid axis is, however, still far from being elucidated, partially due to the limitations of existing tests.
SCREENED aims to develop 3D in vitro tests to characterise the effects of EDCs on thyroid gland function.
SCREENED will deliver highly innovative “Organ-on-a-chip” models, where thyrocyte cells organised in a 3D structure, will be hosted in a microfluidic cell culture device (hereafter called microfluidic bioreactor). This device will mimic the microenvironment of the thyroid gland, by simulating tissue- and organ-level physiology. The first 3D models will consist of thyroid organoids able to recapitulate the thyroid hormone production functionality of the native thyroid (mouse and human models). In addition, we will work on ECM-scaffold-based and on a bioprinted based 3D models, which will represent an even more complex version of the 3D thyroid models, where the “Organ-on-a-chip” will also be supported by a vascularized network.
In period 2, we have improved the protocol to derive thyroid follicles from mouse embryonic stem cells (mESCs). Besides, we have been working in generating a human-derived thyroid tissue using embryonic stem cells (hESCs). Excitingly, our results demonstrate that we can generate, by genetic and chemical manipulation, structures that are capable to organize three-dimensionally in follicles, produce thyroid hormone in vitro, and rescue the levels of TH when transplanted in thyroid-ablated mice. The establishment of a human thyroid follicles from hESCs constitutes a major breakthrough in the field of thyroid research and will be extremely useful to study thyroid gland illnesses beyond EDCs screening.

In parallel, we have successfully developed a microfluidic bioreactor system that is capable of hosting up to eight 3D thyroid tissue constructs in parallel, under flow conditions and that meets the requirements for high throughput screening. We have proved that, in flow conditions, mESC-derived thyroid follicles were able to recapitulate in vivo-like 3D follicular structures, featuring TG expression and a luminal space outlined by ZO-1 expression. Interestingly, follicles cultured in the bioreactor chips revealed an increase in T4 production and storage in the luminal space, as compared to follicles cultured in static conditions. In the same bioreactor platform, we successfully integrated sensors for continuous in situ oxygen measurement. Finally, our bioreactor and battery prototypes for EDCs screening were fabricated and initial experiments revealed that they are capable of providing leak-tight conditions.

We developed few variations of a rat thyroid 3D in vitro model based on 3D collagen scaffolds, 3D Matrigel environments, or decellularized thyroid ECM scaffolds. The rat thyroid progenitor/stem cells that were seeded or encapsulated in these scaffolds showed to be able to repopulate completely the constructs and to self-organize into thyroid follicle morphology able to secrete thyroid hormone. We further developed a 3D bioprinted thyroid construct using a microfluidic bioprinting system. We evaluated the possibility to bioprint not only single cells, but also more complex and physiologically relevant