**4.3. Functional studies**

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 other cultures regarding hormone synthesis.

Follicles' accumulated and organified 125Iradiactivity is determined by well counter. Rat 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 (O) is determined.

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 [82].
