**5.1. The importance of obtaining closed follicles for culturing them**

Many isolated cells and follicular fragments were obtained when thyrocytes became detached after 30 contiguous min, instead of 10 minute enzyme dissociation time (Figure 3A); these thyrocytes were eliminated 24 h later when culture medium was changed. If some fragments persisted after 1 or 2 days' culture they became dissociated and thyrocytes died because they could not adhere to the agarose covered culture support [75] since normal cells require support for growing in culture. Trypan blue allowed an approximation of follicles'

356 Thyroid Hormone

**4.3. Functional studies** 

(O) is determined.

**4.4. Statistical analysis** 

of different doses of iodide and TSH.

[82].

**5. Results** 

other cultures regarding hormone synthesis.

Follicles' accumulated and organified 125I-

antibodies (1:1,000). The nuclei are visualised using DAPI/PBS (1:9,000). Follicles are compressed on commercial laminas as their diameter is greater than that of cells; a 1 mm high chamber was constructed to enable observing by CM without follicular compression.

Iodide organification (which is essential in thyroid hormone synthesis) becomes lost during the first days in the thyroid culture models described to date. Functional analysis of follicles in culture for determining whether follicular architecture is conserved has advantages over

follicles are washed 3 times for 5 min at 50*g* and at 30*g* for pig follicles with COON + Na127I (cold) 100 times the concentration of radioactive iodide used in culture and the radioactivity arising from accumulated (A) is determined. 10% trichloroacetic acid + Na127I 100 times the radioactive iodide concentration used in culture is then added and the radioactivity present in protein precipitate corresponding to protein binding iodide (PBI) or iodide organification

The precipitate is used for determining the amount of DNA by diphenylamine method [88], Tg19S by HPLC and iodine-thyronine (MIT, DIT, T3, rT3 and T4) in Tg by inverse-phase HPLC [89]. The results are expressed in % iodide dose in g DNA. Follicle structure does not allow the number of cells to be counted and statistical analysis requires having a parameter letting the results be homogenised; based on 1 pg DNA/mL equals 2E5 cells, the number of cells present in cultures can be determined and the results statistically correlated

Data given in the text are expressed as mean standard deviation for N values. Significant differences are established between some times for A and O variables by Student's t-test.

Closed follicular architecture is indispensable in agarose culture since open follicles become dissociated and cells die; it is thus essential to begin with maximum closed follicles possible to avoid this. The following item gives the criteria determined important for beginning culture. Then, the long-term culture results in which closed and isolated follicle morphology and function were compared. Last, the morphological and functional results in the presence

Many isolated cells and follicular fragments were obtained when thyrocytes became detached after 30 contiguous min, instead of 10 minute enzyme dissociation time (Figure

**5.1. The importance of obtaining closed follicles for culturing them** 

radiactivity is determined by well counter. Rat

**Figure 3. A.** A follicle fragment from euthyroidic pig thyroid dissociated for 30 min without interruption pre-incubated for 12 h. Note the contour of thyrocytes which became detached from the follicular fragment and cell waste in culture support. **B.** Trypan blue for recently isolated rat thyroid follicles by strong dissociation (Strong dissociation, Table 1). Follicles which did not open (star) conserved colloid birefrigence and a clear and continuous boundary between colloid and cells, whilst those which became resealed (clear arrow) lost colloid birefrigence and the boundary between cavity and cells was not clear. Openings could be seen in those which did not reseal (triangles beam). Cells which did not exclude stain were mainly endothelium cells found in follicle periphery (solid arrows). Follicle fragments presented non-viable thyrocyte aggregates (circle). **C.** Pig euthyroid resealed follicles 12 h pre-incubation. Note that the thyrocytes' apical boundaries could be distinguished due to a lack of colloid. **D.** Trypan blue of closed follicle with colloid birefrigence of pig hypothyroids pre-incubated for 12 h; colloid was birefrigent and the boundary between colloid and thyrocytes was clear and continuous (IM. Scale bar: A 20 μm, B 50 μm, C 15 μm, D 70 μm).

closed or open state (Figure 3B); those conserving colloid showed up due to birefringence in IM and there was a clear boundary between colloid and cells (Figures 3B star and 3D) whilst those which became resealed lost their colloid birefringence and the boundary between cavity and cells was not clear (Figures 3B clear arrow and 3C). Openings in follicles which did not become resealed appeared (Figure 3B triangles beam) as did colloid loss. Cells which did not exclude the stain were mainly endothelium cells which were found on follicle periphery (Figure 3B black arrows). Follicle fragments could be seen because they lost follicular structure and organisation continued from thyrocytes' epithelial layer (Figure 3B circle).

Thyroid Culture from Monolayer to Closed Follicles 359

**Figure 4.** Autoradiograph of dissociated follicles (following pre-incubation, they were added for 2 h in the presence of 5 Ci/mL Na125I). Iodide ions which did not bind to proteins were eliminated while washing the histotechnic preparation until being impregnated in resin. Closed follicles were labelled in the follicle centre even though they became resealed (arrow). **A.** Follicles isolated by strong dissociation (aliquot of follicles from Table 1, strong dissociation with pre-incubation). Follicles seeming to be closed by histological crosssection but which did not accumulate organified iodide in colloid were open follicles (circle). **B.** Most follicles isolated by mild dissociation (aliquot of follicles from Table 1, mild dissociation with pre-incubation) were closed because of intense labelling within follicular cavities (OM. Toluidine bleu. Scale bar: 25 μm).

Closed follicles counted on autoradiographies corresponded to O/A proportion percentage; thus mild dissociation gave 83% closed follicles and 85% O/A proportion whilst strong

Follicles isolated by mild dissociation, following 2 h with radioactive iodide, accumulated iodide 4.8 times and 7.8 times more with 0.1 and 1 mU/mL TSH, respectively, regarding without TSH. TSH did not modify morphology regarding follicular architecture or O/A

This follicle isolation method can be applied to other thyroid tissues from other species, such as rabbits or humans. The percentage of open follicles was greater in pig or human thyroid follicles and more tissue was obtained per experiment; 12 h was thus allowed for pre-

dissociation gave 45% closed and 58% O/A proportion (Table 1).

proportion (Table 2).

incubation (Figures 4C and 4D).

Follicle morphology and function were analysed after 4 hours' labelling with radioactive iodide, varying according to the dissociation procedure used with and without preincubation. If pipette dissociation after each 10 min enzyme digestion was done in such a way that hydrodynamic forces were produced with turbulence, this was called **strong dissociation,** but when this was done slowly without turbulence in the liquid and avoiding air-bubble formation it was called **mild dissociation**.

Iodide which did not bind to molecules was eliminated in fixation liquid until being impregnated in resin; labelling in autoradiographs (following pre-incubation, added for 2 h in the presence of Na125I 5 Ci/mL) was that which bound to proteins and was equivalent to organified iodide. The number of closed follicles could thus be counted and distinguished from open ones by autoradiographs. Open follicles were numerous in strong dissociation; they could be seen because no organified iodide was concentrated within follicle interior (Figure 4A star) even though histological cross-sections seemed to suggest that the cubic epithelial layer continued (Figure 4A circle). There were much fewer open follicles if dissociation was mild (Figure 4B). Follicles which resealed could be distinguished because, even though the labelling was homogeneous, it was less intense in colloid than in those which remained closed (Figure 4B arrow).

Pre-incubation time was another key aspect for obtaining a maximum of closed follicles at the beginning of culture (4 h for rat follicles and 12 h for pig follicles when this was divided into 4 initial hours in which medium was changed by aspirating the medium with follicles and spinning at 30*g* x 5 min followed by pre-incubation for 8 h). The pre-incubation period was essential since this time allowed cells to recover from the aggression of the enzyme used for dissociation.

Pre-incubation time following dissociation promoted an increase in closed follicles with two dissociations (strong or mild, 33% and 46%, respectively, Table 1). Open follicles which did become resealed or follicular fragments (Figure 4A) became disorganised and were discarded in the supernatant when changing the medium and spinning to wash them.

Morphological modifications included modification of the amount of iodide accumulated and organified by follicles (Table 1); if dissociation was mild, iodide accumulation (A) and organification (O) values and O/A percentage increased following pre-incubation time, whereas if dissociation was strong then these values were lower.

circle).

closed or open state (Figure 3B); those conserving colloid showed up due to birefringence in IM and there was a clear boundary between colloid and cells (Figures 3B star and 3D) whilst those which became resealed lost their colloid birefringence and the boundary between cavity and cells was not clear (Figures 3B clear arrow and 3C). Openings in follicles which did not become resealed appeared (Figure 3B triangles beam) as did colloid loss. Cells which did not exclude the stain were mainly endothelium cells which were found on follicle periphery (Figure 3B black arrows). Follicle fragments could be seen because they lost follicular structure and organisation continued from thyrocytes' epithelial layer (Figure 3B

Follicle morphology and function were analysed after 4 hours' labelling with radioactive iodide, varying according to the dissociation procedure used with and without preincubation. If pipette dissociation after each 10 min enzyme digestion was done in such a way that hydrodynamic forces were produced with turbulence, this was called **strong dissociation,** but when this was done slowly without turbulence in the liquid and avoiding

Iodide which did not bind to molecules was eliminated in fixation liquid until being impregnated in resin; labelling in autoradiographs (following pre-incubation, added for 2 h in the presence of Na125I 5 Ci/mL) was that which bound to proteins and was equivalent to organified iodide. The number of closed follicles could thus be counted and distinguished from open ones by autoradiographs. Open follicles were numerous in strong dissociation; they could be seen because no organified iodide was concentrated within follicle interior (Figure 4A star) even though histological cross-sections seemed to suggest that the cubic epithelial layer continued (Figure 4A circle). There were much fewer open follicles if dissociation was mild (Figure 4B). Follicles which resealed could be distinguished because, even though the labelling was homogeneous, it was less intense in colloid than in those

Pre-incubation time was another key aspect for obtaining a maximum of closed follicles at the beginning of culture (4 h for rat follicles and 12 h for pig follicles when this was divided into 4 initial hours in which medium was changed by aspirating the medium with follicles and spinning at 30*g* x 5 min followed by pre-incubation for 8 h). The pre-incubation period was essential since this time allowed cells to recover from the aggression of the enzyme used

Pre-incubation time following dissociation promoted an increase in closed follicles with two dissociations (strong or mild, 33% and 46%, respectively, Table 1). Open follicles which did become resealed or follicular fragments (Figure 4A) became disorganised and were discarded in the supernatant when changing the medium and spinning to wash them.

Morphological modifications included modification of the amount of iodide accumulated and organified by follicles (Table 1); if dissociation was mild, iodide accumulation (A) and organification (O) values and O/A percentage increased following pre-incubation time,

whereas if dissociation was strong then these values were lower.

air-bubble formation it was called **mild dissociation**.

which remained closed (Figure 4B arrow).

for dissociation.

**Figure 4.** Autoradiograph of dissociated follicles (following pre-incubation, they were added for 2 h in the presence of 5 Ci/mL Na125I). Iodide ions which did not bind to proteins were eliminated while washing the histotechnic preparation until being impregnated in resin. Closed follicles were labelled in the follicle centre even though they became resealed (arrow). **A.** Follicles isolated by strong dissociation (aliquot of follicles from Table 1, strong dissociation with pre-incubation). Follicles seeming to be closed by histological crosssection but which did not accumulate organified iodide in colloid were open follicles (circle). **B.** Most follicles isolated by mild dissociation (aliquot of follicles from Table 1, mild dissociation with pre-incubation) were closed because of intense labelling within follicular cavities (OM. Toluidine bleu. Scale bar: 25 μm).

Closed follicles counted on autoradiographies corresponded to O/A proportion percentage; thus mild dissociation gave 83% closed follicles and 85% O/A proportion whilst strong dissociation gave 45% closed and 58% O/A proportion (Table 1).

Follicles isolated by mild dissociation, following 2 h with radioactive iodide, accumulated iodide 4.8 times and 7.8 times more with 0.1 and 1 mU/mL TSH, respectively, regarding without TSH. TSH did not modify morphology regarding follicular architecture or O/A proportion (Table 2).

This follicle isolation method can be applied to other thyroid tissues from other species, such as rabbits or humans. The percentage of open follicles was greater in pig or human thyroid follicles and more tissue was obtained per experiment; 12 h was thus allowed for preincubation (Figures 4C and 4D).


Thyroid Culture from Monolayer to Closed Follicles 361

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

**TSH 0.0 0.1 1.0 0.0 0.1 1.0**

0 57 46 69 91 93 94 1 69 75 71 78 94 93 3 64 85 85 84 86 87 6 0 58 83 60 51 84 9 2 51 71 59 78 91 12 0 54 82 50 73 90

**Exp. 1 Exp. 2**

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:

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)

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

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

accumulation on Tg19S slightly increased with TSH, but not significantly so (Table 4).

Culture, day

and Tg19S content in follicles did not vary.

also became reduced (Table 4).

presence of TSH and remained higher than 80% (Table 3).

**Table 3.** Evolution of 125I-

mU/mL.

**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 preincubation, 125I accumulation (A) and incorporation in proteins (O) were determined following 2 h in the presence of Na125I 5 Ci/mL and expressed in μg/dose/μg DNA.


**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 (these values were representative of 3 experiments).

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.
