**2.3. Isolation and monolayer culture or cell culture**

350 Thyroid Hormone

adopting antagonic roles [34].

**2. Thyroid culture** 

**2.1. Introduction** 

adapts once such 48 h have elapsed and organified iodide escapes, producing new hormones [29]; NIS expression becomes reduced at this time, as does iodide capture [30]. It has been suggested that there is a reduction in the function of the molecules implicated in organification and hormone formation: TPO, Duox 1 and 2, pendrin and Tg [30,31]. Thyrocytes in culture in the presence of 10-E3 M iodide reduce NIS expression and inhibit TPO and Tg synthesis [32]. Such reduction of NIS does not happen in hypothyroid mice, nor is Duox 1 and 2, TPO, pendrin and Tg gene expression modified [33]. An excess of iodide leads to iodide organification inhibition depending on TPO and not on NIS. TSH effects become reduced in the presence of strong concentrations of iodide, resulting in them

Thyroid tissue fragments were kept on glass in saline solution, or *in vitro* (as this involved a glass vessel). Established the neuron cell theory, it has since been established that the unit of life is a cell (i.e. cell theory 1910) and cell culture or *in vitro* study began [35]. Cell cultures were then developed, thereby leading to studying cell functions in controlled conditions and different descriptions of culture mediums, supports and conditions have been developed from 1910. Fibroblasts in culture leave a matrix on culture surface on which endothelium cells from blood capillaries can be cultured; this has been called an extracellular matrix (ECM) [36]. Extracellular supports close to the ECM surrounding cells *in vivo* (such as

New culture techniques were developed in 1975 in view of the close structure-function relationship, recognising the importance of organs' functional units, such as isolating and culturing isles of Langerhans from the pancreas [38] or "acini" regions from the lactant mammary gland or epithelial structures which needed to be conserved in polarised cell

A brief description of the most pertinent techniques for culturing the thyroid, isolated

Organ culture or organotypical culture consists of culturing an organ's fragments or explants. Regarding the thyroid, this began with 2 to 3 h incubations (the term usually used to refer to cultures lasting less than 24h) of sheep thyroid fragments in the presence of radioactive iodide thereby demonstrating *in vitro* the ion's incorpoproportion into diiodinethyronine (DIT) and T4 [40]. When the transmission electron microscope was developed in the 1970s, this led to an ultra-structural description of thyrocytes *in vitro*; the first descriptions of thyrocytes' morphological changes in different culture conditions were made. It was shown that organ culture thyrocytes had reduced RER and GC in the absence of TSH [41,42]. Thyrocyte follicular architecture and ultra-structure were rapidly lost in

culture [39]. Thyroid follicle incubations and cultures could also be mentioned here.

collagen, laminin, fibronectin or matrigel) are currently being used [37].

thyrocytes and/or thyroid follicles is given below.

**2.2. Organotypical culture or organ culture** 

Monolayer culture (better known as cell culture) mainly deals with a single cell type. This implies tissue dissociation by enzymatic digestion or mechanical action and the isolation of cellular types by different sepaproportion methods. Isolated cells are placed on different types of supports where they adhere and proliferate in a single layer until reaching confluence (called primary culture). Secondary culture consists of sowing cells removed from the primary culture in fresh recipients and so on. The term passage is used to indicate the number of successive secondary culture sowings, thus the 1st secondary culture is the 1st cell passage. Thyrocytes were first cultured in 1911 [45]. Using this dissociation technique and continuous shaking during culture has shown that sheep thyrocytes concentrate radioactive iodide and incorporate it in iodine-thyronine: MIT, DIT and T4 [46]. Isolated and small cells, aggregates of 10 to 15 thyrocytes, are obtained after dissociation with trypsin [47,48]. One of the greatest drawbacks is the loss of cultured thyrocytes' cellular polarity when one wishes to study thyroid physiology since such polarity is fundamental in conserving thyrocyte membrane domains, and thus the expression of domain-specific molecules guaranteeing hormone synthesis [49].

Thyrocyte cultures were developed in dual chambers during the 1990s on cubic monolayers as *in vivo* with binding complexes in the lateral membranes' apical region, separating in thyrocytes' the apical membrane domains from the basolateral membrane domains, TSH favouring such cellular polarisation [50]*.* This model has demonstrated that ion flow is determined by thyrocytes' polarity, thereby corroborating the fact that ion channels are different in both thyrocytes membrane domains when thyrocytes' cubic form is conserved. A new channel has been described for the thyrocytes' apical membrane [51]; this new channel is CLC5 which is located in the apical region *in vivo* and it has been proposed that thyrocytes have a position in the apical membrane for controlling pendrin, the I- /Cltransporter [15].

The foregoing has shown the importance of conserving cell polarity and cubic form in thyrocyte culture for studying the gland's physiology and biochemistry.

**Cell lines** are continually growing and indefinitely proliferating cell cultures because they have lost control over their own cell division, contrary to primary and secondary cell cultures which die after a finite number of passes or subcultures, as is genetically determined in normal cells. Thethyrocyte cell line was described [52,53]; Fischer rat thyroid cell line or FRTL is most used around the world as it has a more similar ultra-structure to thyrocytes and synthesised Tg. These have been very useful in studying gene expression, cytoskeleton modification with different factors, iodide flow and that of other ions.

Such studies have led to advances being made in knowledge regarding some precise processes but there are limitations for extrapolating this to the gland *in vivo* because they are cells which lost certain control over their tissue of origin.

Thyroid Culture from Monolayer to Closed Follicles 353

for promoting hormone formation, as happens with thyroid gland apical membrane *in vivo*. It is so important that specific genes have been described which govern follicle formation and maintain follicle architecture, the gene transcribing the thyroid-specific enhancerbinding protein (T/ebp or Nkx2.1) regulating the transcription of genes implicated in hormone synthesis: NIS, TPO, TSH receptor and Tg and re-organised transfecteds thyrocites in follicles [78]. Human goitre follicle structure has been covered with collagen to preserve it longer and cavities in human [79,80] and mouse thyrocyte [78] thin cell bilayers have persisted 2 days more. Collagen's importance in preserving epithelial cells' apical-basal polarity has been described for obtaining MDKC (Madin-Darby canine kidney epithelial cell

line) cell 3D cultures, but hepatic growth factor (HGF) was added to cultures [81].

organification, Tg, T3, T4 synthesis and NIS localisation.

research and care of animals for domestic consumption.

**4.1. Isolation and closed follicle culture** 

is described below.

**4. Method used** 

The methodology developed by our group for reproducibly obtaining closed follicles, conserving their architecture and function in culture analogously to that of the gland *in vivo*

Most of the first work on rat follicle culture was open and became disorganised during the first days of culture [16,23,72]. We based our approach on these rat thyroid dissociation techniques. We describe the importance of isolating closed follicles, their culture and longterm response to TSH and iodide (9 and 12 days) and to increasing doses of iodide. Details are given of the isolation methodology, the morphological study of these isolated follicles and in culture at morphological level by inverted (IM) optical (OM), electron (TEM) and (CM) confocal microscope and their functional study: iodide accumulation and

Wistar rat (200g) and ICR mouse (30g) thyroid was used; the animals were obtained from the Universidad Nacional de Colombia's Bioterium. Pig Cialta and strain 769 thyroid was provided by two slaughterhouses in Bogotá. Some having sub-clinical hyperthyroidism due to - energetic injection, having T3 (1.34 ng/dL) and T4 (107.0 ng/dL) within the normal range and excessively low TSH (<0.005 mUI/mL), were called hypothyroidic, as morphologically and functionally described in mice [33], whilst the others were called euthyroidic. The animals were handled according to Colombian considerations for animals being used in

The methodology mainly involved rat thyroid and was corroborated in mouse thyroid. Obtaining human thyroid fragments is difficult; pig was thus used due to its similarity with human metabolism [82,83,84,85,86], even though it is hoped to begin cultures with human thyroid in the near future. The differences between rodents and pig had to be considered. General metabolism regulated by the thyroid gland in rodents is 10 times greater than that in pigs and humans. Follicle diameter ranges from 50 to 150 μm in rodents, whilst this is 150 to 500 μm in pigs and humans. Rodent lobes range from 3 to 5 mm3 at their widest whilst
