**4.1. Isolation and closed follicle culture**

Wistar rat (200g) and ICR mouse (30g) thyroid was used; the animals were obtained from the Universidad Nacional de Colombia's Bioterium. Pig Cialta and strain 769 thyroid was provided by two slaughterhouses in Bogotá. Some having sub-clinical hyperthyroidism due to - energetic injection, having T3 (1.34 ng/dL) and T4 (107.0 ng/dL) within the normal range and excessively low TSH (<0.005 mUI/mL), were called hypothyroidic, as morphologically and functionally described in mice [33], whilst the others were called euthyroidic. The animals were handled according to Colombian considerations for animals being used in research and care of animals for domestic consumption.

The methodology mainly involved rat thyroid and was corroborated in mouse thyroid. Obtaining human thyroid fragments is difficult; pig was thus used due to its similarity with human metabolism [82,83,84,85,86], even though it is hoped to begin cultures with human thyroid in the near future. The differences between rodents and pig had to be considered. General metabolism regulated by the thyroid gland in rodents is 10 times greater than that in pigs and humans. Follicle diameter ranges from 50 to 150 μm in rodents, whilst this is 150 to 500 μm in pigs and humans. Rodent lobes range from 3 to 5 mm3 at their widest whilst

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.

Thyroid Culture from Monolayer to Closed Follicles 355

Different supports have been tried for maintaining follicular architecture, such as glass, plastic, collagen or collagen coated (1mg/mL), and 1%, 5% and 10% FCS concentration. Closed follicles are conserved in culture; however, thyrocyte monolayers grow proportionally to serum concentration in the medium. Monolayer growth should be avoided because this increases iodide accumulation values and interferes with analysis of iodide organification function; fresh enzyme dissociation must be carried out to recover the follicles [82,86]. When follicular cell fragments are cultured in collagen they do not reform follicles as has been described for human [79,80] and mouse thyrocytes [78]; some MDKC epithelial cells are organised in follicle-like structures requiring HGF [81]. The foregoing meant that the use of glass, plastic and collagen for follicle culture was rejected and agarose type I used

Follicles' functional and morphological state was controlled before beginning the cultures by 5% Trypan blue exclusion exam [87], cell viability was determined by IM which also allowed visualising open or closed follicle architecture. Thyrocyte viability is usually around 100% immediately after isolation and before beginning pre-incubation. Cells which do not exclude Trypan blue are usually endothelium cells bound to follicle periphery (Figure 3B

Iodide accumulation and organification is determined (5 Ci/mL Na125I, 4 h) for ensuring a high percentage of closed follicles; the importance of this control before beginning culture is

Follicles are culture for 0, 1, 3, 6, 9 and 12 days with TSH and without TSH (1 and 0.1 mU/mL rat; 1 mU/mL pig) in the same conditions as for pre-incubation. Culture medium is changed during these days aspirating it with the follicles and spinning at 50*g* for rat and 30 *g* pig, discarding the supernatant which usually contains cell and follicle fragments. The follicles are examined by IM during each stage. 5 Ci/mL Na125I is added each culture day 4

The follicles are cultured for 1 day with and without TSH (0.1 mU/mL rats; 1 mU/mL pigs) for studying the effect of different iodide concentrations. The medium is changed and the follicles cultured for 0.5, 3, 8, 12, 24 and 48 h with 10E-10, 10E-7, 10E-5, 10E-3 M Na127I and Na125I 5 μCi/mL (kindly donated by Manuel E Patarroyo) with and without TSH. The

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,

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

follicles are collected after such treatment for morphological and functional analysis.

solid black arrows) or follicular fragment aggregate thyrocytes (Figure 3B circle).

described and discussed 125I in 6.1. Importance of obtaining closed follicles.

h before collecting the follicles for morphological and functional studies.

autoradiographed slices are observed by OM and ultra-fine slices by TEM.

**4.2. Morphological studies** 

instead.

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

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 and mice and in a third and fourth for pigs.

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

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].

Different supports have been tried for maintaining follicular architecture, such as glass, plastic, collagen or collagen coated (1mg/mL), and 1%, 5% and 10% FCS concentration. Closed follicles are conserved in culture; however, thyrocyte monolayers grow proportionally to serum concentration in the medium. Monolayer growth should be avoided because this increases iodide accumulation values and interferes with analysis of iodide organification function; fresh enzyme dissociation must be carried out to recover the follicles [82,86]. When follicular cell fragments are cultured in collagen they do not reform follicles as has been described for human [79,80] and mouse thyrocytes [78]; some MDKC epithelial cells are organised in follicle-like structures requiring HGF [81]. The foregoing meant that the use of glass, plastic and collagen for follicle culture was rejected and agarose type I used instead.

Follicles' functional and morphological state was controlled before beginning the cultures by 5% Trypan blue exclusion exam [87], cell viability was determined by IM which also allowed visualising open or closed follicle architecture. Thyrocyte viability is usually around 100% immediately after isolation and before beginning pre-incubation. Cells which do not exclude Trypan blue are usually endothelium cells bound to follicle periphery (Figure 3B solid black arrows) or follicular fragment aggregate thyrocytes (Figure 3B circle).

Iodide accumulation and organification is determined (5 Ci/mL Na125I, 4 h) for ensuring a high percentage of closed follicles; the importance of this control before beginning culture is described and discussed 125I in 6.1. Importance of obtaining closed follicles.

Follicles are culture for 0, 1, 3, 6, 9 and 12 days with TSH and without TSH (1 and 0.1 mU/mL rat; 1 mU/mL pig) in the same conditions as for pre-incubation. Culture medium is changed during these days aspirating it with the follicles and spinning at 50*g* for rat and 30 *g* pig, discarding the supernatant which usually contains cell and follicle fragments. The follicles are examined by IM during each stage. 5 Ci/mL Na125I is added each culture day 4 h before collecting the follicles for morphological and functional studies.

The follicles are cultured for 1 day with and without TSH (0.1 mU/mL rats; 1 mU/mL pigs) for studying the effect of different iodide concentrations. The medium is changed and the follicles cultured for 0.5, 3, 8, 12, 24 and 48 h with 10E-10, 10E-7, 10E-5, 10E-3 M Na127I and Na125I 5 μCi/mL (kindly donated by Manuel E Patarroyo) with and without TSH. The follicles are collected after such treatment for morphological and functional analysis.
