**4. Method used**

352 Thyroid Hormone

**2.4. Pseudofollicles** 

**3. Follicles** 

pancreas [67] or kidneys [68] are continued.

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

Some of the main problems involved in thyroid physiology *in vitro* studies are the loss of follicle architecture, thyrocyte polarity and T3 and T4 hormone synthesis [54]. As mentioned above, the follicle lumen disappears in the monolayer with its colloid and the thyrocyte membrane domain polarity necessary for carrying out hormone synthesis [55,56]. However, tridimensional structures can be induced by covering monolayers with ECM elements, they become re-organised into two- to four-cell structures around the intercellular cavity in the presence of TSH, called pseudofollicles by some authors and "follicles" by others [54,56,57,58,59,60,61,62]. These pseudofollicles are unstable, short-duration structures and do not reproduced *in vitro* the function of iodide incorporation in Tg or synthesis of T3 or T4, and shown that follicle structures' thyrocyte polarity is necessary for studying thyroid

A new culture technique was developed from 1965 to 1980 where the functional unit of different epithelial organs, isles of Langerhans in the pancreas [64,65] or the acinar region of

It is clear that conserving multicellular structures *in vitro* forming exocrine glands' functional units depends on preserving apical-basal polarity for imitating *in vivo* functions; this is why attempts at characterising the factors generating polarity and which molecules allow maintaining 3D structures *in vitro* in mammary gland acini [66], the endocrine

The 1980s saw the beginning of 24-h thyroid follicle cultures [23,69,70,71,72,73,74] on agarose, to avoid cell adhesion and monolayer formation [75]. Such incubations showed that iodide organification and H2O2 production took place in colloid [23,72] and that rat thyrocitos of open and closed follicles incubated for 12 h on agarose conserved their apicalbasal polarity and *in vivo* ultra-structure, in basal region nucleus surrounded by RER, supranuclear GC, in apical region vesicles' and microvellosities. As well as responding to TSH-forming pseudopods [16,23,75], synthetic TSH peptides bound in the basolateral membrane [76]. Closed follicles were maintained for up to 3 days [69] and responded to thyrocytes' TSH, increasing RER [23]. TSH has stimulated pig and human open follicle thyrocyte function in culture during the first two days [49,74]. Pig open follicles cultured for 48 h with forskolin have been used for determining the function of H2O2 formation which is important in hormone synthesis [77]. The presence of TSH- or forskolin-induced cyclic adenosine monophosphate (AMPc) route stimulants has been seen to be indispensable in these cultures and has reiterated the importance of conserving follicle structure in culture

the mammary gland [39] are isolated and cultivated, conserving *in vivo* morphology.

physiology and the molecular processes implicated in such function [63].

cells which lost certain control over their tissue of origin.

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