*5.2.2. Morphological study*

Pig hypothyroid follicles cultured 1 and 3 days without TSH have a very thin epithelial layer (Figure 5A) like original tissue's follicle epithelium. The epithelium became cubic after day 6 and was preserved up to day 9 (Figure 5C). It was seen that follicles conserving colloid had a birefrigent aspect when observed by IM, thereby showing that this was conserved during culture. Epithelium thickness increased in the presence of TSH, becoming cubic on the first day of culture (Figure 5B) and being maintained so until day 9.

#### Thyroid Culture from Monolayer to Closed Follicles 365

364 Thyroid Hormone

It has been described that pig thyroid mini organ cultures enables studying "thyroid tissue structural and functional integrity *in vitro* [54]"; however, we have considered that studying thyroids *in vitro* is better done with isolated follicles than using mini organ cultures. Since closed follicles maintain their architecture throughout culture time, thyrocytes are viable and their basement membrane is in direct contact with the medium and not with capillaries

DNA content per culture dish did not show a significant change during 12 days' rat follicle culture (1.55 ± 0.52 g/dish, N = 16) or 9 days' pig follicle culture (5.35 ± 0.36 g/dish, N = 25).

Even though closed and isolated follicles in culture had differences regarding stable Tg19S, the amount of T3 and T4 and iodide accumulation between different treatments with and without TSH and with or without 10E-10 M NaI, the follicles did have more iodide organification, iodised Tg19S and T3 and T4 at 12 days' culture, even without TSH [83,86], than in all other culture models published to date except of the group [83,84,85,86]. Such

Our culture system has different characteristics distinguishing it from other models described up to now. Monolayers lose their function on the first day [48], become reorganised in pseudofollicles on the third day and only 2% to 4 % become incorporated or organified in iodine accumulated in poorly iodised Tg (Tg16S) [91], even though higher than 90% O/A with TSH has been reported for a matrigel-covered monolayer culture forming a double cell layer having cavities [61]. Different models mentioning culturing "follicles" [92,93,94,95] have not shown these functions in their results; others culturing pig "follicles" for 2 days, based on Björkman and Ekholm [16] as we, require 5% FCS, 1 mU/mL TSH and non-physiological molecules such as forskolin or 8-(4-chlorophenylthio)-cAMP for maintaining thyrocyte functions [77]. Using closed follicles enables functional parameters to be conserved and measured: iodide accumulation, iodide organification and, particularly, Tg19S equivalent to that *in vivo* and T3 and T4 formation throughout culture with or without TSH. We have also shown that maintaining closed follicular architecture is an indispensable condition for conserving such thyroid functions in culture *in vitro*. If follicular architecture is to be conserved, it is not enough to maintain functions at the same values as those at the start of culture as TSH is required and culture becomes improved by adding iodide, as

Pig hypothyroid follicles cultured 1 and 3 days without TSH have a very thin epithelial layer (Figure 5A) like original tissue's follicle epithelium. The epithelium became cubic after day 6 and was preserved up to day 9 (Figure 5C). It was seen that follicles conserving colloid had a birefrigent aspect when observed by IM, thereby showing that this was conserved during culture. Epithelium thickness increased in the presence of TSH, becoming cubic on

whose endothelial cells die rapidly in culture during the first 24 h (Figure CB).

variations were homologous to glands *in vivo* in the same study conditions.

thyroid function *in vivo* is governed by TSH and iodide.

the first day of culture (Figure 5B) and being maintained so until day 9.

*5.2.2. Morphological study* 

**Figure 5.** Morphological aspect of follicles in culture. A. Hypothyroid follicle 1 day's culture without TSH; birefringent colloid and thin follicle epithelium can be seen. **B.** Hypothyroid follicle 1 day's culture with 1mU/mL TSH; cubic epithelium and thyrocytes' apical poles typical of a resealed follicle can be seen. **C.** Hypothyroid follicles in 9 days' culture without TSH; the epithelium is cubic and colloid birefringence can be seen in all follicles. **D**. Euthyroid follicle 1 day culture without TSH. A resealed follicle (circle) having irregular contour between thyrocytes and cavity can be seen in the centre. Closed follicles preserve colloid birefringence and regular boundary between thyrocytes and colloid since the start of culture. **E** Pig euthyroid follicles in the presence of 1 mU/mL TSH; follicular cavities are difficult to distinguish. **F.** Autoradiography of rat follicles cultured 12 days in the presence of TSH (1 mU/mL) corresponding to experiment 2, Table 3. Follicle cavities are evident due to the organified iodide found only in very narrow follicular cavities (Scale bar: A and D 50 μm, B 25 μm, C 130 μm, E 100 μm, IM. F 150 μm, OM. Toluidina bleu).

Rat or pig euthyroid follicles without TSH kept the same follicular architecture throughout the whole culture time (Figure 5D). Colloidal cavities became reduced from the third day in the presence of TSH and were difficult to distinguish on day 9 and 12 by IM (Figure 5E); however, they could be seen by autoradiography where only iodide bound to molecules could be identified and they were only located in follicles' very narrow colloidal cavities (Figure 5F). Colloidal cavities' boundaries could also be seen by labelling thyrocytes' apical membrane protein SLC5A8 (short-chain fatty acids transporter) [84].

Rat thyrocyte and pig euthyroid follicle ultra-structure in **one day** culture without TSH (Figure 6A) and with TSH (Figure 6B) was comparable to that for cells *in vivo* (Figure 1C). They conserved their polarity and organelles, but exocytic vesicles were difficult to distinguish from those from endocytosis. RER was more abundant in the presence of TSH and microvellosities were more evident than without TSH.

Thyrocytes had vacuolated and reduced RER and GC following **3 days**' culture without TSH; the GC could be seen in supra-nuclear position (Figure 6C G) as could numerous autophagic vacuoles (Figure 6C arrow) and secondary lysosomes. This became modified in

Thyroid Culture from Monolayer to Closed Follicles 367

the presence of TSH, presenting abundant RER and GC (Figure 6D G) and containing more autophagic vacuoles and secondary lysosomes in thyrocytes' apical region (Figure 6D arrows) than without TSH. RER and GC became more reduced by the **sixth day** without TSH and thyrocytes became thin. Whilst autophagic vacuoles and secondary lysosomes

Thyrocytes were thin by **day 9** and **12** without TSH and had exiguous RER and GC and reduced microvellosities. The nuclei contained very little heterochromatin (Figure 6E). Follicular centres were very narrow in the presence of TSH, thyrocytes had abundant GC and RER reaching the cells' apical regions (Figure 6F) and the other organelles had the same

Adding 10E-10 M NaI did not modify thyrocytes' follicular architecture or ultra-structure;

Pig thyrocyte and hypothyroid follicle ultra-structure had exiguous RER and GC when culture began (Figure 7A), like original gland hypothyroids *in vivo*, and culture (even without TSH) developed these organelles from the third day of culture [86]. These hypothyroid follicles' thyrocytes became cubic in the presence of TSH (Figure 5B) from the first day of culture and it was seen that the RER and GC developed and became more evident on the **sixth** and **ninth day** of culture (Figure 7B), similar to rat or pig euthyroid

Thyrocytes had normal mitochondria in rat follicle and pig euthyroidic and hypothyroid

**Figure 7.** Pig hypothyroid follicle culture. **A.** RER and GC were exiguous and cells were thin 1 day in the absence of TSH. **B.** 9 days in the presence of 1 mU/mL TSH. Cross-section of thyrocyte follicle basal

Similar culture models to these have been described. Pig follicle culture has highlighted the importance of thyrocyte polarity, but OM morphological study was limited on day 1 [74,94]. Another, dealing with "normal" human follicle culture of thyroidectomy for goitre requiring

pole. Abundant RER can be seen around the nucleus (TEM. A 7,800 X, B 9,000 X).

became reduced with TSH, RER and GC also did so by day 3.

however, RER and GC were preserved up to day 12 without TSH.

follicle response with TSH, but follicular cavity did not become reduced.

distribution as on the first days of culture.

cultures for all culture times.

**Figure 6.** Ultra-structure of thyrocytes from follicles cultured without TSH A 1 day, C 3 days, D 12 days and with 1 mU/mL TSH B 1 day, D 3 days and F 12 days. Binding complexes were preserved in thyrocytes' lateral membrane apical region during all the times with and without TSH. **A.** Thyrocytes conserved their polarisation; microvellosities in contact with electron-dense colloid. Binding complexes were located in lateral membrane's apical region between cells. The rugose endoplasmic reticulum (RER) was found to be slightly vesiculated. **B.** Thyrocyte ultra-structure was comparable to without TSH, even though RER was more abundant. **C. and D.** Ultra-structure was conserved in thyrocytes; the supranuclear Golgi complex (G) and more abundant RER in thyrocytes in the presence of TSH can be seen. Autophagic vacuoles (C. arrow) and secondary lysosomes (D, arrows) with or without TSH can be seen. **E.** Thyrocyte polarity was conserved. Colloid was electron-dense and separated from follicle exterior. Thyrocytes had exiguous RER and G. **F.** Organelles were well conserved. RER was well developed and occupied thyrocytes' apical region (TEM. A and C 7,300 X, B 7,500 X, D 7,438 X, E 9,810 X, F 8,260 X).

the presence of TSH, presenting abundant RER and GC (Figure 6D G) and containing more autophagic vacuoles and secondary lysosomes in thyrocytes' apical region (Figure 6D arrows) than without TSH. RER and GC became more reduced by the **sixth day** without TSH and thyrocytes became thin. Whilst autophagic vacuoles and secondary lysosomes became reduced with TSH, RER and GC also did so by day 3.

366 Thyroid Hormone

**Figure 6.** Ultra-structure of thyrocytes from follicles cultured without TSH A 1 day, C 3 days, D 12 days and with 1 mU/mL TSH B 1 day, D 3 days and F 12 days. Binding complexes were preserved in thyrocytes' lateral membrane apical region during all the times with and without TSH. **A.** Thyrocytes conserved their polarisation; microvellosities in contact with electron-dense colloid. Binding complexes were located in lateral membrane's apical region between cells. The rugose endoplasmic reticulum (RER) was found to be slightly vesiculated. **B.** Thyrocyte ultra-structure was comparable to without TSH, even though RER was more abundant. **C. and D.** Ultra-structure was conserved in thyrocytes; the supranuclear Golgi complex (G) and more abundant RER in thyrocytes in the presence of TSH can be seen. Autophagic vacuoles (C. arrow) and secondary lysosomes (D, arrows) with or without TSH can be seen. **E.** Thyrocyte polarity was conserved. Colloid was electron-dense and separated from follicle exterior. Thyrocytes had exiguous RER and G. **F.** Organelles were well conserved. RER was well developed and occupied thyrocytes' apical region

(TEM. A and C 7,300 X, B 7,500 X, D 7,438 X, E 9,810 X, F 8,260 X).

Thyrocytes were thin by **day 9** and **12** without TSH and had exiguous RER and GC and reduced microvellosities. The nuclei contained very little heterochromatin (Figure 6E). Follicular centres were very narrow in the presence of TSH, thyrocytes had abundant GC and RER reaching the cells' apical regions (Figure 6F) and the other organelles had the same distribution as on the first days of culture.

Adding 10E-10 M NaI did not modify thyrocytes' follicular architecture or ultra-structure; however, RER and GC were preserved up to day 12 without TSH.

Pig thyrocyte and hypothyroid follicle ultra-structure had exiguous RER and GC when culture began (Figure 7A), like original gland hypothyroids *in vivo*, and culture (even without TSH) developed these organelles from the third day of culture [86]. These hypothyroid follicles' thyrocytes became cubic in the presence of TSH (Figure 5B) from the first day of culture and it was seen that the RER and GC developed and became more evident on the **sixth** and **ninth day** of culture (Figure 7B), similar to rat or pig euthyroid follicle response with TSH, but follicular cavity did not become reduced.

Thyrocytes had normal mitochondria in rat follicle and pig euthyroidic and hypothyroid cultures for all culture times.

**Figure 7.** Pig hypothyroid follicle culture. **A.** RER and GC were exiguous and cells were thin 1 day in the absence of TSH. **B.** 9 days in the presence of 1 mU/mL TSH. Cross-section of thyrocyte follicle basal pole. Abundant RER can be seen around the nucleus (TEM. A 7,800 X, B 9,000 X).

Similar culture models to these have been described. Pig follicle culture has highlighted the importance of thyrocyte polarity, but OM morphological study was limited on day 1 [74,94]. Another, dealing with "normal" human follicle culture of thyroidectomy for goitre requiring

TSH [93] had a TEM image showing thyrocytes having the ultra-structure for cells in the process of cell death with lysed mitochondria, without RER or GC. Using this human follicle culture model enabled analyzing the effect of TSH [95] or cytokines [95,96] without presenting culture morphology.

Thyroid Culture from Monolayer to Closed Follicles 369

**% O/A M, NaI 10E-10 10E-7 10E-5 10E-3** 

0.5 82 64 0 0 1 84 42 0 0 2 88 90 5 0 3 92 88 3 0 4 95 87 6 0 5 88 89 6 0 6 84 93 4 0 **Table 7.** Response of organification percentage regarding iodide accumulation (O/A proportion) by rat follicles cultured in the presence of increasing doses of iodide (NaI, M) and Na125I 5 Ci/mL. The follicles were cultured on agarose with 0.5% FCS for 1 day, medium was changed and culture began with NaI and TSH experimental points. Each value was the average of two samples or culture dishes

Iodide accumulation and organification in rat follicles could be assimilated to biochemical reactions after 30 min with and without TSH and thus define a constant (Km) and maximum speed (Vmax). Accumulation apparent Km without TSH was 5x10E-6 M and 10E-7 M de NaI with TSH; iodide organification apparent Km was 5x10E-7 M and was not modified by the presence of TSH (Figure 8). Iodide accumulation by isolated follicles corresponded well with saturable iodide transport characteristics [15,99] and stimulated TSH thereby reducing Km but not Vmax (Figure 8). It should be stated that organification had to be inhibited and

iodide accumulation (**A**) and organification (**B**) for follicles in culture measured

following half an hour in the presence of increasing doses of 127-I Na and 5 Ci/mL 125-I Na expressed in log M with and without TSH. The follicles were cultured on agarose with 0.5% FCS for 1 day, the medium was changed and culture began with NaI and TSH experimental points. The results were the

average of two samples and were expressed as percentage of maximum value.

Culture, hours

expressed in %/dose/μg DNA.

**Figure 8.** Initial 125I-

Our results showed that if culture was begun with closed follicles then extracellular matrix support elements were not required [56,57,79,80] nor was TSH for maintaining follicular cavity, as has been described in most pseudofollicle or follicle cultures reconstructed from monolayers, or similar structures called "follicles" [57,50,60,61,62,79,96], thereby demonstrating that if closed follicles are used from the start of culture they conserve their morphology, having the correct polarity as that of their thyrocytes in culture and have a binding complex (tight junction, belt desmosome and spot desmosome) in lateral membranes' apical part as well as *in vivo* [9]. Such closed follicles in culture responded to TSH, like other *in vitro* models [42] or like gland follicles in normal *in vivo* to TSH stimulus [3,25], epithelium thickness, RER and GC becoming increased, and follicular cavities becoming notably reduced [11,83,90]. Follicle response to the absence of TSH both *in vivo*  [24] and *in vitro* [42] was also comparable as these organelles became reduced. This effect became reverted *in vivo* when TSH was added, reactivating thyroid functions [11,33,97].

Pig closed and isolated hypothyroid follicles behaved like the gland *in vivo* when the hypothyroid effect was deleted [24], RER and GC increased, follicular epithelium became thin to cubic and culture time became faster in the presence of TSH, but follicular cavity did not become reduced during the 9 days of culture.

Our results thus showed that long-term thyroid follicle function and morphology can be maintained *in vitro,* being equivalent to the gland *in vivo*.

The next section describes the effect of increasing doses of iodide on closed follicles in culture.
