Context:Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid-cancer lines were found to be heavily contaminated by other sources and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that loss of thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture (5H), including hormones with marked differences in affinity for the relevant rodent/human receptor.Objective:To define conditions which allows proliferation of primary human follicular thyrocytes for many passages without losing phenotype.Methods:Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by STRs. Human-rodent inter-species contamination was assessed.Results:We defined an 'h7H medium' enabling growth of human thyroid cultures for more than twenty passages maintaining thyrocyte phenotype. Thyrocytes proliferated and grouped as follicle-like structures (FLS); expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin and thyroperoxidase, showed iodine-uptake and secreted thyroglobulin and FT3. Using these conditions, we generated a Bank of Thyroid Tumors in Culture (BANTTIC) from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas) and carcinomas.Conclusions:Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The BANTTIC generated under humanized h7H culture conditions will provide a much needed tool to compare similarly growing cells from normal versus pathological origins, and thus to elucidate molecular basis of thyroid disease.