**3. Follicles**

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

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

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 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].

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* is described below.
