**5.2. Long-term closed follicle culture**

### *5.2.1. Functional study*

On **day zero** (4 h pre-incubation in the presence of radioactive iodide) follicles accumulated 3.5 ± 2.1% of the dose in the medium **without TSH** (9 experiments in rats). Organified iodide percentage varied according to closed follicle percentage at the start of each experiment. O/Aprioportion was 57% in experiment 1 and 91% in experiment 2 (Table 3); more than two thirds of organified iodide bound to stable Tg19S (Table 4), the rest of the molecules having lower molecular weight. Capture increased in the presence of **TSH** regarding without TSH 250 ± 210% (9 experiments in rats), but O/A percentage remained constant. The effect of TSH was evident on A and O, and higher than values without TSH (Tables 2, 3 and 4). Iodide accumulation on Tg19S slightly increased with TSH, but not significantly so (Table 4).

360 Thyroid Hormone

incubation, 125I-

**Without pre-incubation**

**With pre-incubation 4 hours**

**Digestion conditions % follicles closes A O % O/A** 

Mild dissociation 57 ± 1,4 1.14 ± 0,01 0.89 ± 0,01 78 Strong dissociation 45 ± 5,7 1.84 ± 0,37 1.08 ± 0,14 58

Mild dissociation 83 ± 1,4 1.93 ± 0,24 1.64 ± 0,17 85 Strong dissociation 60 ± 7,1 1.32 ± 0,17 0.91 ± 0,01 68 **Table 1.** Influence of dissociation conditions and pre-incubation time on the percentage of rat thyroid isolated and closed follicles and their function. It can be seen that the percentage of organified iodide on accumulated iodide (O/A) was equivalent to the percentage of closed follicles concentrating radioactive ion determined by follicle count using accumulated grains in follicular colloid in autoradiography of semi-fine cross-sections (Figure 4). Average values for two culture dishes ± SD. Following pre-

the presence of Na125I 5 Ci/mL and expressed in μg/dose/μg DNA.

(these values were representative of 3 experiments).

**5.2. Long-term closed follicle culture** 

*5.2.1. Functional study* 

accumulation (A) and incorporation in proteins (O) were determined following 2 h in

**TSH A O % O/A**  None 0.73 ± 0,18 0.58 ± 0,11 79 0,1mU/mL 3.48 ± 0,17 2.78 ± 0,04 80 1mU/mL 5.71 ± 0,72 4.98 ± 0,67 87 **Table 2.** Influence of TSH on iodide accumulation and organification in isolated rat follicles expressed in μg/dose/μg DNA. Follicles were cultured for 2 h in the presence of Na125I 5Ci/mL immediately following pre-incubation. TSH was stimulated by Na125I but O/A proportion was around 80% with or without TSH. The value without TSH represented the percentage of closed follicles obtained following correct mechanical dissociation and pre-incubation of follicles. Average values for culture dishes ± SD

A good approximation of the percentage of closed follicles following pre-incubation must thus be born in mind (this corresponded to day 0 in our cultures). It can thus be generalised that O/A proportion values should be greater than 80% before beginning culture (day 0, Table 2) and that there should be a potentially high number of closed follicles, even though follicle diameter may vary in each species or come from different thyroid functional states [85,86,90]. Open follicles' ability to reveal themselves during the course of pre-incubation probably depended on the degree of initial opening. Even though groupings of rat thyroid cells became organised in the presence of TSH and on agarose, they became reorganised into 6- to 10-cell follicles which could be cultured for 3 days [69,73]. In our results, rat or pig follicles which became resealed did not require TSH for conserving their follicular architecture.

On **day zero** (4 h pre-incubation in the presence of radioactive iodide) follicles accumulated 3.5 ± 2.1% of the dose in the medium **without TSH** (9 experiments in rats). Organified iodide percentage varied according to closed follicle percentage at the start of each experiment.


**Table 3.** Evolution of 125I organification/accumulation in rat follicle culture. Comparing two experiments where rat closed follicle percentage varied during the course of two experiments (Exp. 1 and Exp. 2). 80% of follicles were closed at the start of culture counted in autoradiographs of culture aliquots in experiment 2 (*cf* 65% in experiment 1). Na125I 5 Ci/mL was added 4 h before collecting follicles during each day of the experiment, expressed in %/dose/μg DNA. Each value was the average of two samples or culture dishes. Experiment 2 was representative of 7 independent experiments. TSH: mU/mL.

Following **one day**'s culture without TSH, accumulation was low at the start of culture; it did not become modified, but if it was high it became reduced, whilst incorpoproportion of iodide in Tg became reduced (Table 4). T3 and T4 proportion in regarding Tg was not modified. Iodide accumulation increased in the presence of TSH, O/A percentage remained high and in starting values (Table 3). Iodide incorpoproportion in Tg19S was better (Table 4) and Tg19S content in follicles did not vary.

Accumulation fell abruptly on the **third day** without TSH when starting with a low percentage of closed follicles, just like O/A percentage, whilst it became reduced with a high percentage of closed follicles, but O/A was maintained. O/A values were high in the presence of TSH and remained higher than 80% (Table 3).

Accumulation and organification values and their O/A percentage differed on the **sixth day** regarding closed follicle percentage; A and O could not be determined in experiment 1 which began with 57% (Table 3. Exp. 1) whilst experiment 2 began with 94% and became reduced to 60% (Table 3. Exp. 2). There was a reduction in all O/A percentages in rat or pig cultures by the sixth day which also differed with the percentage of closed follicles (Table 5), animal species and functional state at the start of culture. The percentage of iodide on Tg19S also became reduced (Table 4).


Thyroid Culture from Monolayer to Closed Follicles 363

**Euthyroid Hipothyroid**

0 43.4. ±.5.9 4 82.4. ±.7.2 4 1mU/mL 61.5. ±.4.6 4 80.5. ±.3.7 4

0 56.94. ±.4.7 3 77.0. ±.3.8 4 1mU/mL 71.4. ±.7.1 2 76.44. ±.3.9 4

0 37.4. ±.6.6 2 82.24. ±.4.1 3 1mU/mL 43.2. ±.5.9 2 76.44. ±.5.3 2

0 9.16. ±.2.2 2 23.3. ±.9.4 2 1mU/mL 15.0. ±.3.7 2 52.32. ±.4.2 2

0 52.66. ±.2.2 2 16.62. ±.1.5 3 1mU/mL 64.8. ±.4.3 2 53.6. ±.5.0 3

organification/accumulation (O/A proportion) percentage for pig follicles.

**Culture, day TSH % O/A N % O/A N** 

Hypothyroid follicles had high O/A percentage at the start regarding euthyroids (representative results from 4 experiments). This experiment began with less than 50% O/A in euthyroids for comparing with Table 3, experiment 1; however, when 3 experiments began with euthyroid follicles having 80% or more O/A they behaved like those for rat follicles in experiment 2, Table 3. 5 Ci/mL Na125I were added 4 h before collecting follicles on each day of the experiment expressed in %/dose/μg DNA. Average values

The difference between euthyroidic rat or pig follicles regarding hypothyroid ones was that hypothyroids responded to TSH on the sixth and ninth culture days, perhaps due to adapting to normal culture conditions, like hypothyroid glands' response *in vivo* when the

We compared isolated follicles' iodide accumulation and organification with their respective pig mini organ cultures lasting up to 9 days. Euthyroidic tissue became disorganised after the sixth day and functionality could not be determined after this day. Some follicles were conserved in the hypothyroids on the outside of the cultured fragment and presented this function on days 6 and 9 (Table 6). Mini organ culture functional values were higher than those of follicles isolated on the first day, but these were not significant; they were higher from the first day onwards in isolated follicles, maintaining higher values up to day 9

**TSH A O % A/O N TSH A O % A/O N** 

0 0.049 0.025 51.6 ± 0.052 4 0 CND CND 3 1mU/mL 0.074 0.048 65.3 ± 0.043 4 1mU/mL CND CND 3

0 0.122 0.0633 36.75 ± 0.67 4 0 0,115 0.025 22.7 ± 1.5 3 1mU/mL 0.122 0.0655 53.6± 0.050 4 1mU/mL 0.723 0.278 38.5 ± 0.067 2 **Table 6.** Determining iodide accumulation (A) and organification (O) for isolated follicles and mini organ cultures from the same pig thyroids after 9 days' culture. 5 Ci/mL Na125I was added 4 h before collecting follicles and mini organ cultures on each day of the experiment expressed in %/dose/μg DNA. Average g of follicle DNA was 4.5 and 14.7 for mini organ cultures. Values represent the average

**Euthyroid, follicles Euthyroid, mini organ culture** 

**Hypothyroid, follicles Hypothyroid, mini organ culture** 

effect induced by hypothyroidism stimulated with TSH was eliminated [24].

0

1

3

6

9

**Table 5.** Evolution of 125I-

for the number (N) of culture dishes ± SD.

(euthyroidic and hypothyroid) (Table 6).

number (N) of culture dishes ± SD. CND: could not be determined.

**Table 4.** Evolution of iodide accumulation (A) and its incorporation in stable Tg19S in rat follicle culture. Effect of adding a small dose of cold iodide (10E-10 M Na127I). A was expressed in %/dose/μg DNA. Radioactivity determined in stable Tg19S was expressed in % 125I accumulation/dose/μg DNA. The results without NaI were those expressed in experiment 2, Table 3. Na125I 5 Ci/mL 4 h was added before collecting follicles during each day of the experiment. Each value was the average of two samples or culture dishes.

O/A retained the same values on **day 9 and 12** as those for day 6 in both experiments without TSH, whilst with TSH this increased in experiment 1 regressed to day 1 values and then maintained similar values to those of the two first days. The percentage of iodide on Tg19S with and without TSH was conserved (Table 4).

The presence of a small dose of iodide (10E-10 M NaI) did not modify iodide accumulation values at the start of culture (Table 4), but responded better to TSH. Tg19S had more T3 and T4 and TSH increased the percentage of T3. O/A percentage increased with 0.1mU/mL TSH on days 6, 9 and 12 whilst it fell with 1 mU/mL (not significantly). Iodide had no effect on stable Tg19S content in follicles; more than two thirds of accumulated iodide was incorporated in Tg19S (Table 4) even in the absence of TSH where iodide accumulation was very low on day 12.

Preserving the function of pig follicle thyrocytes during culture also depended on the percentage of closed follicles since the beginning. However, this differed from those for rats in the absence of TSH, because this began with 43% O/A equivalent to the percentage of closed follicles presenting iodide organification on days 6 and 9 (Table 5, euthyroidic follicles) contrary to rat follicles which was zero (Table 3. Exp. 1). As 7 times more material was obtained from pig thyroids than rat follicles, a greater number of recovered follicles were conserved in changes of medium by centrifuging, or different species having variations in response to the same medium conditions.


**Culture. Day Without NaI** 

**With NaI** 

very low on day 12.

Effect of adding a small dose of cold iodide (10E-

response to the same medium conditions.

Radioactivity determined in stable Tg19S was expressed in % 125I-

Tg19S with and without TSH was conserved (Table 4).

**A 125I- in Tg19S\***

10 M Na127I). A was expressed in %/dose/μg DNA.

accumulation/dose/μg DNA. The results

**TSH 0.0 0.1 1.0 0.0 0.1 1.0** 

0 5.8 5.5 7.2 59 63 67 1 1.4 2.7 3.1 43 66 65 3 1.3 4.5 8.7 46 50 52 6 0.7 1.2 4.0 25 13 42 9 0.2 0.6 4.0 16 17 55 12 0.2 0.7 8.0 15 25 52

0 5.8 7.8 9.3 60 61 66 1 1.1 1.8 3.1 31 58 64 3 0.7 4.2 7.6 49 49 52 6 0.1 1.4 3.4 10 22 39 9 0.2 1.6 2.6 14 32 38 12 0.1 1.9 2.9 11 33 58 **Table 4.** Evolution of iodide accumulation (A) and its incorporation in stable Tg19S in rat follicle culture.

without NaI were those expressed in experiment 2, Table 3. Na125I 5 Ci/mL 4 h was added before collecting follicles during each day of the experiment. Each value was the average of two samples or culture dishes.

O/A retained the same values on **day 9 and 12** as those for day 6 in both experiments without TSH, whilst with TSH this increased in experiment 1 regressed to day 1 values and then maintained similar values to those of the two first days. The percentage of iodide on

The presence of a small dose of iodide (10E-10 M NaI) did not modify iodide accumulation values at the start of culture (Table 4), but responded better to TSH. Tg19S had more T3 and T4 and TSH increased the percentage of T3. O/A percentage increased with 0.1mU/mL TSH on days 6, 9 and 12 whilst it fell with 1 mU/mL (not significantly). Iodide had no effect on stable Tg19S content in follicles; more than two thirds of accumulated iodide was incorporated in Tg19S (Table 4) even in the absence of TSH where iodide accumulation was

Preserving the function of pig follicle thyrocytes during culture also depended on the percentage of closed follicles since the beginning. However, this differed from those for rats in the absence of TSH, because this began with 43% O/A equivalent to the percentage of closed follicles presenting iodide organification on days 6 and 9 (Table 5, euthyroidic follicles) contrary to rat follicles which was zero (Table 3. Exp. 1). As 7 times more material was obtained from pig thyroids than rat follicles, a greater number of recovered follicles were conserved in changes of medium by centrifuging, or different species having variations in **Table 5.** Evolution of 125I organification/accumulation (O/A proportion) percentage for pig follicles. Hypothyroid follicles had high O/A percentage at the start regarding euthyroids (representative results from 4 experiments). This experiment began with less than 50% O/A in euthyroids for comparing with Table 3, experiment 1; however, when 3 experiments began with euthyroid follicles having 80% or more O/A they behaved like those for rat follicles in experiment 2, Table 3. 5 Ci/mL Na125I were added 4 h before collecting follicles on each day of the experiment expressed in %/dose/μg DNA. Average values for the number (N) of culture dishes ± SD.

The difference between euthyroidic rat or pig follicles regarding hypothyroid ones was that hypothyroids responded to TSH on the sixth and ninth culture days, perhaps due to adapting to normal culture conditions, like hypothyroid glands' response *in vivo* when the effect induced by hypothyroidism stimulated with TSH was eliminated [24].

We compared isolated follicles' iodide accumulation and organification with their respective pig mini organ cultures lasting up to 9 days. Euthyroidic tissue became disorganised after the sixth day and functionality could not be determined after this day. Some follicles were conserved in the hypothyroids on the outside of the cultured fragment and presented this function on days 6 and 9 (Table 6). Mini organ culture functional values were higher than those of follicles isolated on the first day, but these were not significant; they were higher from the first day onwards in isolated follicles, maintaining higher values up to day 9 (euthyroidic and hypothyroid) (Table 6).


**Table 6.** Determining iodide accumulation (A) and organification (O) for isolated follicles and mini organ cultures from the same pig thyroids after 9 days' culture. 5 Ci/mL Na125I was added 4 h before collecting follicles and mini organ cultures on each day of the experiment expressed in %/dose/μg DNA. Average g of follicle DNA was 4.5 and 14.7 for mini organ cultures. Values represent the average number (N) of culture dishes ± SD. CND: could not be determined.

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

Thyroid Culture from Monolayer to Closed Follicles 365

**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

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

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

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

membrane protein SLC5A8 (short-chain fatty acids transporter) [84].

and microvellosities were more evident than without TSH.

150 μm, OM. Toluidina bleu).

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

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