Human Thyroid Cultures Research Articles

Humanized Medium (h7H) Allows Long-Term Primary Follicular Thyroid Cultures From Human Normal Thyroid, Benign Neoplasm, and Cancer

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 the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor. The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype. Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed. We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas. Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.

Effect of GLP1R agonists taspoglutide and liraglutide on primary thyroid C-cells from rodent and man

Glucagon-like peptide 1 (GLP1) analogs have been associated with an increased incidence of thyroid C-cell hyperplasia and tumors in rodents. This effect may be due to a GLP1 receptor (GLP1R)-dependent mechanism. As the expression of GLP1R is much lower in primates than in rodents, the described C-cell proliferative lesions may not be relevant to man. Here, we aimed to establish primary thyroid cell cultures of rat and human to evaluate the expression and function of GLP1R in C-cells. In our experiments, GLP1R expression was observed in primary rat C-cells (in situ hybridization) but was not detected in primary human C-cells (mRNA and protein levels). The functional response of the cultures to the stimulation with GLP1R agonists is an indirect measure of the presence of functional receptor. Liraglutide and taspoglutide elicited a modest increase in calcitonin release and in calcitonin expression in rat primary thyroid cultures. Contrarily, no functional response to GLP1R agonists was observed in human thyroid cultures, despite the presence of few calcitonin-positive C-cells. Thus, the lack of functional response of the human cultures adds to the weight of evidence indicating that healthy human C-cells have very low levels or completely lack GLP1R. In summary, our results support the hypothesis that the GLP1R agonist-induced C-cell responses in rodents may not be relevant to primates. In addition, the established cell culture method represents a useful tool to study the physiological and/or pathological roles of GLP1 and GLP1R agonists on normal, non-transformed primary C-cells from rats and man.

The effect of basic fibroblast growth factor on the growth and function of human thyrocytes

Basic fibroblast growth factor (bFGF) was quantitated in human primary thyrocyte cultures and thyroid cell lines produced by transfection with pSV3neo. Immunoreactive-bFGF (ir-bFGF) bound to heparin-Sepharose affinity columns eluted with 1.8-2.0 mol NaCl/l and had a molecular weight of approximately 17,000. Recombinant human bFGF in the presence of 5% serum increased the growth of transfected human thyrocytes but not the growth of primary human thyrocytes. Preincubation of cells with up to 100 micrograms bFGF/l potentiated TSH-stimulated cAMP release from the transfected cells but inhibited release from primary human thyroid cultures. bFGF may be an important modulator of thyroid cell function and growth.

Short- and long-term evaluation of normal and abnormal human thyroid cells in monolayer culture.

We have investigated the TSH responsiveness of normal and abnormal human thyroid cells cultured in the short term with high serum concentrations and for up to 6 months in a low serum, chemically-defined, medium. Cells from normal human thyroid tissue (n = 9), multinodular goitre (n = 6), benign follicular adenomata (n = 6), and differentiated thyroid carcinoma (n = 3) formed confluent monolayers which were sensitive to bovine TSH (bTSH) in concentrations greater than 25 microU/ml when assessed by the intracellular response of cyclic AMP at 7 d of culture. Such sensitivity was less than that observed with a continuously proliferating thyroid cell line (FRTL-5) derived from Fisher rat thyroid and which responded to concentrations of bTSH as low as 5-10 microU/ml. Human cells derived from iodine/antithyroid drug treated Graves' thyroid tissue (n = 6) were less sensitive than normal cells requiring up to 500 microU/ml bTSH to increase intracellular cyclic AMP and poorly differentiated thyroid cancer cells (n = 3) failed to respond to bTSH. Long-term human thyroid cultures of normal and follicular adenoma cells in the chemically-defined medium used for the FRTL-5 cells had absent fibroblast growth and continued in monolayer form without significant follicle formation. These cells remained highly sensitive to bTSH stimulation when tested after 4, 13, and 26 weeks of continuous culture. All such cell preparations failed to proliferate under conditions which favoured the rapid growth of the rat thyroid cells. These data demonstrated that while thyroid cell culture conditions described in the literature do not permit proliferation of human thyroid cells, they do allow an assessment of their functional state in vitro which may lead to a further understanding of thyroid cell pathophysiology.

Neuropeptidergic control of cyclic AMP accumulation in human thyroid cell

Somatostatin (SRIF), cholecystokinin (CCK), gastrin and substance P, as single agents, do not influence baseline cellular cAMP levels in human thyroid cultures. SRIF inhibits TSH-induced cMAP accumulation in human thyroid cell, while CCK, gastrin and substance P do not modify cAMP response to TSH. Vasoactive intestinal peptide (VIP) increases cellular cAMP levels in human thyroid cultures and its effect is additive to increases produced by norepinephrine (NE) and isoproterenol (ISO). Neither SRIF nor the other tested peptides influence adrenergic and VIP-ergic cAMP stimulation.

Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture.

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell-surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.

Laboratory studies on the varicella-zoster virus.

Summary In a study of varicella-zoster virus, one strain isolated from a patient with varicella and one strain from a patient with zoster were grown in a range of different cell cultures. The viruses grew in human amnion, human thyroid, human embryo lung, HeLa, rhesus monkey kidney, vervet monkey kidney and V3A cultures. They behaved identically, giving similar cytopathic changes in the same range of cell cultures. Neither virus could be readily obtained in a cell-free state. The isolation of varicella-zoster viruses in cell cultures was accomplished from vesicle fluids of 15 patients with chickenpox and one patient with zoster, and from lung tissue of a fatal case of chickenpox and a fatal case of zoster. Virus was not isolated from the urine of 23 patients with chickenpox and one patient with zoster, nor from the ganglia of the fifth cranial nerve of 66 elderly persons who had died from causes other than chickenpox and zoster. Both viruses in cell cultures were stored satisfactorily for several years at either -65° or -184°C and human thyroid cultures were the most suitable cultures for their subsequent recovery.

Growth of foot-and mouth disease virus in monolayer cultures of calf thyroid cells.

THE most successful laboratory systems for the growth and assay of foot-and-mouth disease virus (FMDV) have been suckling mice1, cell cultures of calf and pig kidney2,3, and the cell line derived from baby hamster kidney (BHK 21)4. All these systems vary in their relative sensitivity for various strains of virus and none appears to effect an absolute determination of the titre of infective virus. Recently it has been found that, in the assay of unmodified cattle virus, higher titres are obtained in primary monolayer cultures of calf thyroid cells than in the systems enumerated and in some instances these cultures are 100–1,000-fold more sensitive. Calf thyroid cultures have been used previously in the isolation and study of the virus of malignant catarrhal fever5 and human thyroid cultures for the isolation of human entero-viruses and adenoviruses6,7.

Characteristics of Herpes Zoster and Varicella Viruses Propagated in Vitro

Summary The agents of varicella and herpes zoster grown in cultures of human amnion retain infectivity only when associated with viable cells. Conditions which lead to cell death as measured by the eosin Y dye exclusion test result in concomitant loss of infectivity. Using a plaque assay method without agar overlay, the optimum conditions for virus absorption, propagation and release have been investigated. The preparation of cell-free infectious varicella virus from infected primary human thyroid cultures has been confirmed.

Die Isolierung von Poliovirus in verschiedenen Gewebekulturen

100 stool extracts from patients with poliomyelitis or from individuals after oral poliomyelitis vaccination were inoculated simultaneously into HeLa, monkey kidney, and human thyroid cultures. In most cases (88 times) a cytopathic effect was seen in all three cultures. Failures were most frequently encountered in thyroid, rarely in monkey kidney cells. They were due either to thyroid culture lots with reduced viral susceptibility or to small amounts of virus in the stool specimens. No qualitative difference was found in the host range of individual virus strains. With attenuated viruses the cytopathic effect in HeLa cells as a rule was delayed for several days.