Reorganization of porcine thyroid cells into functional follicles in a chemically defined, serum- and thyrotropin-free medium.

Abstract

In the serum-free, chemically defined medium NCTC 109, freshly isolated porcine thyroid cells aggregate and form functional follicles in culture even in the absence of thyrotropin. The follicular pattern observed under light and electron microscopy express the main morphological characteristics of in vivo thyroid cells. Follicles are large, replete with dense colloid, and the apical pole of cells is characterized by well-developed microvilli and the presence of aminopeptidase N. The index of iodide transport activity (' 25 1-C/M ratio) decreases vs . days of culture to a resting value of about 1 or 2 at day 2. Addition of thyrotropin (200 pU/ ml final concentration) at day 4 is followed by a 10-fold increase in iodide transport activity within 24 h and a 40-fold increase 4 d later. Incorporation and organification of iodide are dose dependent between 0 and 250 ,uU/ml thyrotropin; highest concentrations (4,000-16,000 p,U/ ml) are significantly inhibitory . In the absence of thyrotropin each cell synthesizes 8.2 pg thyroglobulin/d . Acute stimulation by thyrotropin at day 4 resulted in a slight decrease in the quantity of thyroglobulin present in the cell layer but in an increase in the total amount of thyroglobulin recovered in both cells and medium, reaching 34 .3 pg/cell/d . The protein exported into the medium is thyroglobulin, as shown by SDS PAGE and immunological properties . Here we demonstrate that porcine thyroid cells can be maintained in culture as resting, highly differentiated, follicular-associated cells, sensitive to acute stimulation by thyrotropin.A model system of thyroid cells in culture able to reproduce the main characteristics of the thyroid gland should resemble in vivo follicles with respect to expression of morphological differentiation offollicle-associated cells (26), expression of the specific thyroid metabolism leading to synthesis of thyroglobulin (Tgb) and thyroid hormones (43), and simulation of the events following the interaction between thyroid cells and their physiological stimulator, thyrotropin (TSH) (9) .On the basis of light microscope examination, the reorganization of isolated TSH-stimulated cells into follicle-like structures has been reported in primary cultures of thyroid tissue from sheep (36), lamb (34), hog (13,14,32), steer (59), dog (65), and man (8) The metabolic properties of thyroid cells in vitro have been studied in a general fashion by the same authors . In primary culture, a rapid decrease in thyroxine (T4) production (29), Tgb synthesis (32, 52), and iodide binding (37) are observed. The loss of these activities was retarded but not prevented by exposure of cells to TSH (37). However, in the presence of TSH, porcine thyroid cells actively trap iodide (17), synthesize Tgb (13,32,41), and produce T4 (41) after several days in culture . (For a review, see Lissitzky et al. [421).Some regulatory events relating to TSH action have also been described in support-anchored thyroid cell culture systems and in isolated follicles in suspension. Acute effects...

Thyrocytes, but not C cells, actively undergo growth and folliculogenesis at the periphery of thyroid tissue fragments in three-dimensional collagen gel culture

“…But it has not yet been clearly determined whether thyroid folliculogenesis is TSH independent (Hilfer and Searls 1980;Fayet et al 1982) or TSH dependent (Kerkof et al 1964;Fayet et al 1971). As described in our present study, TSH did not affect thyroid folliculogenesis itself.…”

Long-term iodination of thyroglobulin by porcine thyroid cells cultured in porous-bottomed culture chambers: regulation by thyrotrophin

“…In previous reports, the same TSH concen¬ tration was sufficient to allow iodide uptake and iodination by follicles embedded in collagen gel (Chambard, Verrier, Gabrion & Mauchamp, 1984) or FRTL-5 lines (Marcocci, Luini, Santisteban & Grollman, 1987). Other studies, using different culture systems, reported that TSH stimulated iodide uptake and its incorporation into thyroglobulin but that maxi¬ mal responses were often obtained with TSH concen¬ trations as high as 100 pU/ml (Mauchamp et al 1979) or 200 pU/ml (Fayet et al 1982). This paper presents the first results of an in-vitro study of thyroglobulin iodination using an original cell culture system.…”

“…Short-term iodide uptake and thyroglobulin iodin¬ ation have been performed using different culture systems (Mauchamp, Charrier, Takasu et al 1979;Fayet, Hovsepian, Dickson & Lissitzky, 1982;Chambard et al 1983) where cells cultured for less than 15 days were used. However, in neither system was the iodine content of thyroglobulin reported.…”

Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium

“…they incorporate iodine into organic molecules. However, these follicles lack a basal lamina (Fayet et al, 1982). Without TSH stimulation, thyroid cells can form follicles and a basal lamina if they are cultured in collagen gel (Kitajima et al, 1987;Toda & Sugihara, 1990) or on the surface of a reconstituted basement membrane matrix (Espanet et al, 1992) The mechanism of thyroid cell re-aggregation is still unclear (Toda & Sugihara, 1990;Toda et al, 1993).…”