Human thyroid microtissue assay workflow. A, The microtissue model evaluation consisted of 3 control treatment (Tx) groups: (Tx-1, negative control) baseline effects in hTMT medium without TSH supplementation, (Tx-2, positive control) hTMT medium supplemented with TSH, and (Tx-3, TSH antagonist control) hTMT medium supplemented with TSH and human anti-TSHR K1-70 antibody. B, Human thyrocytes were thawed and seeded directly from cryopreservation into a 3D culture model on 96-well plates with control treatments at 2 day intervals. Microtissue morphology, analyte sampling (TG, T4, T3), and viability were assessed at day 14. C, Standard assay conditions utilized Tx-2 medium changes for the duration of the assay. Reference chemical and solvent control (0.5% DMSO) exposures were initiated on day 8 and repeated every 2 days for a total exposure duration of 6 days. The assay terminated on day 14 where microtissue morphology, analyte sampling (T4), and viability were assessed. Thyroglobulin (TG), thyroxine (T4), triiodothyronine (T3).