develop 3D in vitro cellular assays able to mimic the microenvironment of a thyroid gland at different levels of anatomical complexity:
– An adult thyroid stem cells from rodent origin or human induced pluripotent stem cells (hiPSCs) forming a 3D organoid
– A decellularized scaffold able to reproduce the biological composition of a native thyroid,
which is repopulated by the above-mentioned 3D organoids
– A bioprinted construct comprising the 3D organoids
able to mimic the spatial and geometrical architecture of a native thyroid
develop a modular microbioreactor able to host the three different 3D in vitro assays, with improved culture conditions by perfusing nutrients and low dose ENDOCRINE DISRUPTORS in a controlled manner
produce “magnetic cells” using new superparamagnetic biocompatible particles and use these magnetic cells to support precise spatiotemporal homing within the decellularized and bioprinted constructs
evaluate these 3D in vitro assays, individually and in comparison with each other and with in vivo assays, in a unique sex-specific approach
identify the Mode of action and develop related Adverse outcome pathways of tested EDs on thyroid stem cells / progenitors, leading to
information on Endocrine disruptors capacity to interfere with thyroid development and function
identify at least one biomarker of either mature thyrocytes or thyroid stem/progenitor cells able to reflect the
interference of a chemical product on thyroid function or stem cells capacity to differentiate
engage discussion with all relevant stakeholder groups to ensure the developments are fit for purpose for ED
safety assessment, and achieve a level B classification from OECD
Modular Microbioreactor Development
The aim objective of this Work Package is to develop a modular bioreactor platform based on microfluidic principles with a hierarchical configuration, which allows the analysis of compounds and their effect on engineered thyroid tissue.
The precise control of low volume samples in microfluidics will enable us to efficiently test compounds at very low concentrations under dynamic cell culture conditions. Integrated sensors will provide the possibility of a continuous quality control of the culture conditions and the engineered tissues.
Decellularized ECM Scaffolds
The aim of this Work Package is the preparation and characterization of 3D decellularized rodent thyroid lobe matrices (from young adult, male and female rats), and their capacity to act as 3D biomimetic scaffolds for lobar recellularization with rodent, adult thyroid stem cells/progenitors (from young adult, male and female rats) induced to thyrocyte differentiation.
Recellularization will be achieved using either dynamic cell culture or microbioreactor technology up to reconstruction of a complete and functional, rat 3D thyroid lobe in vitro.
Comprehensive Unbiased and Targeted Proteomic and Genomic Analysis
The aims of this Work Package are to:
– Apply comprehensive gene and protein discovery ‘omics approaches to identify,
develop assays and deliver tests for protein/genes that are modulated by Endocrine Disruptors in the in vitro thyroid assays being developed here, and
– Incorporate the ‘omics data into a comprehensive computer model of thyroid hormone synthesis and distribution to simulate the effects of Endocrine Disruptors on thyroid hormone synthesis in vivo.
The unbiased nature of omics gene and protein analysis combined with a systematic evaluation of the existing Endocrine Disruptors knowledge space will enable us to compare the responses of thyroid cells maintained in the various configurations used here to known EDs.
Rodent and Human Organoid Development
The objectives here are to efficiently produce mouse and human 3D functional Thyroid organoids from pluripotent stem
cells for studying thyroid function in vitro:
– Production of purified functional thyroid follicles from mouse pluripotent stem cells
– Production of purified functional thyroid follicles from human pluripotent stem cells.
Bioprinted Thyroid Model
We will develop a bioprinted 3D in vitro model that will be used to screen the selected EDs in comparison with organoids and decellularized ECM. To develop such a model, we will first screen a selected set of hydrogels to assess the best chemistry to maintain high cell viability of thyrocytes. In parallel, a method to create a perfusable vasculature network will be developed to integrate such vasculature within the bioprinted model. Cells will be magnetized to further control their spatial arrangement within the bioprinted construct to mimic the native anatomical deposition in the thyroid. Finally, the bioprinted model will be characterized in terms of functional thyroid markers by conventional immunohistochemistry and biochemical assays and proteogenomics and integrated in the microbioreactor platform developed in the project.
Evaluation of reliability and sensitivity of the 3D in vitro assays and comparison with in vivo studies
The aim of Work Package is to study the response of the thyroid function to 4 different classes of endocrine-disrupting chemicals (EDs), namely polycyclic aromatic hydrocarbons (PAH), phthalates (PH), organophosphate flame retardants (FR), and polychlorinated byphenyls (PCB). All the molecules chosen have well established interfering action with TH biosynthesis and/or secretion. Two different thyroid function assays will be used:
-In vitro assays: sex-dependent (male and female) thyroid bioconstructs
-In vivo models: pregnant dams and young adult (1 month’s old) male and female rats.
Dissemination, Exploitation, and Stakeholder Interaction
The aim of this Work Package is to disseminate, communicate and exploit the results generated in SCREENED in the most efficient way and to foster sustainability. This will include engaging a dialogue with the main stakeholders that will enable the future uptake of SCREENED results, namely regulatory authorities, industry and policy-makers.