Merck

Functional model of rat thyroid follicles cultured in Matrigel.

Endocrine connections (2021-04-29)
Antonina Khoruzhenko, Françoise Miot, Claude Massart, Jacqueline Van Sande, Jacques Emile Dumont, Renaud Beauwens, Alain Boom
ABSTRACT

Long-term maintenance of functional activity of thyroid cells is an essential requirement for basic in vitro studies on the physiology and pathology of the thyroid. An important prerequisite of thyrocytes' functional activity in vivo and in vitro is their follicle organization. This study aimed at developing a method of cultivation of functionally active rat thyroid follicles in Matrigel under three-dimensional conditions. Undamaged rat thyroid follicles were isolated by enzymatic digestion with collagenase/dispase, then embedded into Matrigel, and cultivated for 2 weeks. Thyroglobulin, thyroxine and zonula occludens-1 (ZO-1) localization were revealed by immunofluorescence analysis. Iodide organification was tested by protein-bound 125I (PBI) measurement. Integrity of the follicles was preserved during the whole period of cultivation and was confirmed by 3D reconstruction of ZO-1 localization. Thyroglobulin was detected in the thyrocyte cytoplasm, as well as in the intrafollicular lumen. Thyroxine was observed predominantly at the apical side of thyrocytes. Also, generated cultures were characterized by a high level of iodide organification: PB125I represented 39% of the total radioactivity in the Matrigel drop embedding the follicles; at the same time, methimazole almost totally inhibited this process (0.2% of total radioactivity). The method of rat thyrocyte cultivation in Matrigel, as described here allows to maintain the structural integrity and the functional activity of thyroid follicles in vitro and could be used for wide ranges of basic and applied researches in thyroidology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dispase® II, protease
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
DAPI, dilactate, ≥98% (HPLC)
Sigma-Aldrich
1,4-Diazabicyclo[2.2.2]octane, ReagentPlus®, ≥99%
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Thyrotropic hormone from bovine pituitary
Sigma-Aldrich
L-Ascorbic acid, powder, suitable for cell culture, γ-irradiated
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
Sigma-Aldrich
Insulin solution from bovine pancreas, 10 mg/mL insulin in 25  mM HEPES, pH 8.2, BioReagent, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Agarose, BioReagent, for molecular biology, low EEO