Abstract
We performed preparation and cell culture of human thyrocytes from biopsies of cold nodules and surrounding tissues. Different methods of primary thyrocyte culture were compared: A) maintenance in RPMI-1640 with 10% FCS until experiment and B) RPMI-1640 with 1% FCS for 24h and subsequent serum withdrawal. Method A induces proliferation in primary thyrocytes, gives a high yield of cells and allows passaging. However, these cells show a dedifferentiated morphology and form a non-organized cell monolayer. In contrast, method B gives a lower yield and does not allow passaging, but the cells remain organized in a follicle-like structure resembling the functional unit of the thyroid. To further compare these culturing methods and the consequences of different cellular organizations on thyroid cell physiology, we assessed the expression of thyroid marker genes upon TSH stimulation using semiquantitative real-time PCR. Moreover, we stimulated TSH-pretreated or untreated thyrocytes with ATP. This leads to an activation of endogenous P2Y receptors and elicits a strong intracellular calcium release measured by calcium imaging. Our results show that the follicular organization achieved with method B results in a higher TSH-mediated thyroid marker gene expression, underlining the importance of cellular organization and its influence on cellular function. Furthermore it allows the examination of spatial and temporal differences of calcium signalling events elicited by TSH which cannot be seen using method A due to the lack of cellular organization. We conclude that method B is better suited to examine TSH-mediated events in thyrocytes.