Endocrine Abstracts

Please Development and preliminary characterization of a simple human thyroid organoid in vitro model for thyroid metabolism investigation For decades, in vitro studies of the thyroid have relied on thyroid-derived tumor cell lines, which are limited by their undifferentiated phenotype, lack of response to TSH, and chromosomal abnormalities. A viable alternative are monolayer (2D) cultures of primary thyrocytes isolated from histologically normal thyroid glands. However, these cultures are short-lived and tend to lose thyroid differentiation status. Furthermore, they cannot recapitulate the structural architecture on which follicular units are dependent for thyroid hormone biosynthesis. Thyrocytes are arranged in a single layer around the colloid. To overcome 2D culture limitations, we developed and characterized a three-dimensional (3D) thyroid culture, comparing it to 2D cultures. Non-tumorous thyroid samples were obtained from healthy donors (University Hospital of Pisa, Italy). The fragmented tissues were digested through collagenase IV (1 mg/ mL)-CaCl₂ at 37°C and 5% CO₂ for 2 hours. The cells were seeded into T-25 flasks in Humanized 7 homeostatic additives (h7H) medium. Then, 80 % confluent primary thyrocytes (P0) were collected and sed in Nunclon Sphera™ 96-well Microplates (10000 cells/cm²) in h7H and 3% Geltrex™. Cell aggregates were collected after 7 to 10 days. Morphology was characterized by transmission electron microscopy (TEM). The expression of functional thyroid markers TPO (Thyroperoxidase), TSHR (Thyroid-Stimulating Receptor), PAX8 (Paired Box-8), TTF-1 (Thyroid Transcription Factor-1), NIS (Sodium/iodide symporter), IYD (Iodotyrosine deiodinase) and TG (Thyroglobulin) was examined by quantitative RT-PCR, immunocytofluorescent staining, western blotting and ELISA. Confocal and TEM analyses revealed a follicle-like 3D structure with the colloid compartment. TPO, TSHR, PAX8, NIS, and TG (P < 0.05) gene expression was significantly higher as compared to 2D cultures. In addition, ELISA test also revealed a higher production of TG protein. In conclusion, our findings revealed that the developed 3D thyroid cultures possessed the morphological traits and peculiarities of the real tissue, both in functionality and marker expression and may therefore be a suitable tool to investigate thyroid metabolism and physiology.