Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function

Abstract

Nilsson M, Husmark J, Nilsson B, Tisell L-E, Ericson LE. Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function. Eur J Endocrinol 1996;135:469–80. ISSN 0804–4643 Epithelial properties of thyrocytes are difficult to maintain in conventional cell culture systems. We used bicameral chambers (Transwell™) in attempts to establish a functional epithelium of thyrocytes of human origin. Thyroid follicle segments were isolated by collagenase digestion of paradenomatous tissue obtained at surgery for follicular adenoma and of tissue from glands with Graves' disease. After careful separation from connective tissue and single cells by centrifugation, the follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) containing 10% fetal calf serum (FCS) or Coon's modified Hams medium enriched with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were converted into a confluent cell layer, which had similar DNA content irrespective of type of medium, after 4–6 days. Cells grown in EMEM or 5H established a transepithelial electrical resistance (R) of 200–500Ω·cm2 and was impermeable to [3H]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially cultured in EMEM or 5H caused a further increase of R, maximally to 1500 Ω·cm2, along with a rise of the transepithelial potential difference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [3H]leucine-labeled thyroglobulin into the apical medium. Cells from Graves' thyroid tissue established an epithelium on the filter with similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a dendritic shape. HLA-DR expression was generally absent in EMEM- or 5H-grown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after interferon-gamma stimulation for 48 h. We conclude that human thyrocytes form a tight and polarized epithelium when cultured on permeable filters. The polarized structure and function of the cells are positively regulated by TSH. The culture system may be useful in studies addressing the role of the epithelial phenotype (cell polarity and tight barrier) in normal thyroid function as well as in pathological processes in the thyroid, such as autoimmunity, cell transformation and tumor progression. Mikael Nilsson, Institute of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3, S-413 90 Göteborg, Sweden