1. Human thyroid glands were obtained at surgery from multinodular goiters.

2. The tissue was incubated in 0.1% type II collagenase (Worthington) overnight at room temperature.

3. The digested tissue was passed through a 200-μm filter to remove undigested material, and the collagenase was removed by centrifugation at 360 g at 4°C for 10 min.

4. The supernatant was discarded, and the cellular pellet was resuspended in DMEM supplemented with 10% newborn bovine calf serum (NCS) containing 100 U/ml penicillin and 100 μg/ml streptomycin.

5. Cells were plated on dishes coated with 1% gelatin. Cells were incubated for up to 1 h and examined microscopically every 10 min.

6. When thyroid follicles began to attach to the dishes, the cell suspension was removed, and the dishes were washed extensively and vigorously with Hanks' balanced salt solution (HBSS) to remove adherent follicles.

7. Single cells remaining adherent after these washings were incubated with fresh endothelial cell medium that consisted of a 1:1 mixture of 10% NCS in DMEM and conditioned media from JEG cells supplemented with 1 ng/ml FGF-2, 17.5 U/ml heparin, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 μg/ml nystatin.

8. JEG cells are a human cell line derived from a choriocarcinoma and are cultured in DMEM containing 10% FBS.

9. Cultures were incubated in humidified incubators with 5% CO^₂-95% air, and the media were replaced every 2 days.

10. The human thyroid endothelial cells used for the experiments reported in this article were obtained from an individual with multinodular goiter who was euthyroid.

References
1. Folkman J, Haudenschild CC, Zetter BR. Long-term culture of capillary endothelial cells. Proc Natl Acad Sci USA. 1979; 76: 5217–5221.

2. Vimal A. Patel, Ann Logan, John C. Watkinson, Saad Uz-Zaman, Michael C. Sheppard, James D. Ramsden, and Margaret C. Eggo. Isolation and characterization of human thyroid endothelial cells. AJP-Endo 2003; 284: E168-E176.