BACKGROUND: Cell models are key instruments for in vitro studies of the thyroid. Permanent thyroid cell lines that are widely used in laboratory research typically originate from tumors. For many purposes it is desirable to compare tumor cells with cells originating from normal tissue. However, such cultures grow slowly, have a highly limited lifespan and are known to lose their thyroid characteristics. The aim of the present study was to type coding and noncoding thyroid markers in different culture systems in an attempt to determine the optimal conditions for in vitro experimentation. METHODS: Human primary thyroid cells were isolated from histologically nontumorous tissues. Two alternative media (6H and H7h) were used. The morphology and behavior of the ensuing monolayer (2-dimensional) cultures was monitored by microscopy. The expression of key thyroid-related genes (n=9) was monitored by RT-PCR on Days 8, 21 and 43 after initiation. As a pilot study the same markers were studied in a 3-dimensional hanging-drop culture system. RESULTS: In the cultures with 6H or H7h medium, the primary thyroid cells displayed growth in numbers and size. Most cells retained the main morphological characteristics of thyroid cells throughout the first two weeks of culture and fibroblast-like cells appeared around Day 19. By Day 21, most thyroid gene markers were retained but by Day 43 several markers were no longer present. The lncRNA transcripts PTCSC2 (spliced) and PTCSC3 were the first to disappear. There were no fundamental differences between the two media in the early period of culture. In the 3-dimensional system most thyroid markers were retained by Day 21. CONCLUSION: Cultures of thyroid cells retain many thyroid characteristics up to Day 21 whereafter fibroblast-like dedifferentiated cells begin to dominate.