**4.2. Morphological studies**

354 Thyroid Hormone

this is 3 to 5 cm3 in pigs and humans, meaning that many rodents must be sacrificed for each experiment; 30% to 40% of a pig's lobe is used. Rodents' capsule is thin and the parenchyma does not have large amounts of connective tissue; this capsule is thick in pigs and connective tissue septa are very abundant and extensive. Shaving razors are used for stereoscopic micro-dissection. The capsule of rat and mice lobes is eliminated; each is cut in two along its major axis whilst pig lobes are opened in two with a scalpel and cut into 7 to 10 mm3 fragments. Connective tissue is then eliminated as far as possible using stereoscopic microdissection with razors without affecting the parenchyma. Around 3 mm3 fragments are obtained (similar to rodent fragments) without connective tissue visible by stereoscope. The thyroid fragments are put together and washed 3 times with COON medium [55], enzymatically dissociated with collagenase II which digests collagen (250 U/mL rodents; 400 U/mL pig) and 2 g/mL DNase 1 (nb, the original article [82] read "2 mg/ml DNase I" when it should have been 2 μg/ml). Dead cells form aggregates which are avoided with DNase which only dead cells' DNA digest and become fragmented; live follicle cells or fragments

Thyroid fragments become dissociated in enzyme solution in COON medium at 37ºC and being shaken at 140 oscillations per minute; without delay, they are mechanically dissociated in this solution, aspirating and expelling enzyme solution containing thyroid fragments with 20 mL pipettes (3 to 5mm distal diameter; extreme for liquid entry and exit from pipettes), 10 times. The technique with rodent fragments continues with 10mL pipettes (1.5 mm distal diameter), 10 times. Such pipette dissociation is done at 10 min intervals during enzyme dissociation (i.e. the supernatant containing isolated follicles is collected every 10 min after pipette dissociation and fresh enzyme solution added for the following 10 min). This is done three times x 10 min with rodent thyroid and 6 x 10 min with pig thyroid; this is a modification of already described dissociation [16,23,72] and is most important for avoiding follicle opening. Most follicles are isolated during a second dissociation for rats

The follicles isolated during each 10 min interval are washed 3 times with COON + 2% foetal calf serum (FCS) spun at 50*g* for rodent follicles and 30*g* for pig follicles. This must be done in a free rotor centrifuge using low centrifugal force, otherwise centrifugal pressure opens up many follicles. All the follicles are placed together and filtered through 100 m pore diameter mesh for rodents; those for pigs are left to decant at 1*g* for 10 min. Dissociation preincubation or recuperation time is continued for 4 h for rats and mice and 12 h for pigs in COON medium + 0.5% FCS in a 95% air - 5% CO2 atmosphere and 100% humidity on 1% agarose type I (less grouping than with agarose type II) to avoid cells adhering to the support [44,75]. Culture medium (the same as pre-incubation) is changed for aspiration with follicles; it is spun at 50*g* for rodents and 30*g* for pigs. The supernatant is skimmed off and fresh medium added to begin culture in the same ambient conditions and on agarose type I;

Undissociated fragments (around 0.8 mm3) remaining after enzyme dissociation are washed 3 times with COON + 2 % FCS and cultured in the same conditions as for follicles but with

2% FCS. This has been called mini organ culture, according to Bauer and Herzog [43].

become attached to these aggregates if DNase is not added [23].

the same is done for changing medium when making the culture.

and mice and in a third and fourth for pigs.

Morphological study involves impregnating follicles in Epon resin; follicles are spun at 300*g*  for dehydratation before being impregnated in the resin [23,82,83]. Semi-fine, autoradiographed slices are observed by OM and ultra-fine slices by TEM.

Protein synthesis and NIS expression reduce excess iodide [30,31,32] and (bearing in mind that NIS has not been described in closed follicle cultures) the presence of NIS is determined in culture in the presence of different iodide and TSH concentrations, with anti-NIS/GS antibodies (1:500, kindly donated by Thierry Pourcher) and Alexa 488 anti-rabbit secondary

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.

Thyroid Culture from Monolayer to Closed Follicles 357

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'

**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).
