E. Rogers1, K. Wolf2, T. Stone2, C. Deisenroth3, J. Chen1, J.-L. Klein1 and E. LeCluyse2
1.LifeNet Health LifeSciences, Virginia Beach, VA, 2. Research Triangle Park, NC, 3. U.S. Environmental Protection Agency, Research Triangle Park, NC
Background and Purpose:
The European Chemicals Agency (ECHA) and the European Food Safety Authority (EFSA) have jointly published guidance for identifying endocrine disruptors. Together with the U.S. EPA’s Endocrine Disruptor Screening Program (EDSP), these initiatives emphasize the need for high-throughput screening (HTS) approaches to efficiently evaluate potential thyroid-disrupting chemicals (TDCs) and minimize reliance on traditional, low-throughput, animal-based methods. Deisenroth et al. previously developed a novel 3D human thyrocyte microtissue platform to identify TDCs that act through various mechanisms relevant to thyroid hormone synthesis. This study was designed to evaluate the within-laboratory reproducibility and performance of the assay using primary human thyrocytes from one donor lot, and to assess the applicability of the 3D model for characterizing responses to known TDCs using thyrocytes from a different single donor.
Methods:
Thyrocytes were derived from healthy donors (≤55 years, BMI ≤35) and cultured in a 96-well 3D format. Control groups included baseline (CT-1), TSHR agonism (CT-2), and TSHR antagonism (CT-3) conditions to assess model sensitivity and specificity. Cells were maintained for 14 days with medium changes every 2 days. Culture media were collected on day 14 for T3 and T4 quantification by ELISA, and microtissue viability was determined using the CellTiter-Glo® assay. Model performance was evaluated by dynamic range, precision, and screening quality metrics. For evaluation of the model with known TDCs, medium samples were collected on day 14 after a 6-day exposure (0.001-100µM) for T4 quantification by ELISA. The half-maximal inhibitory concentration (IC50) for each compound was calculated using GraphPad Prism. Results: The thyrocyte lots met all specification criteria for screening applications. Microtissues were formed within 4 days and evenly distributed. Microtissues produced T4 concentrations exceeding 1.0 ng/mL at day 14, with robust dynamic ranges (rS/B >2.5-fold), coefficients of variation <30%, and Z′-factors >0.6 for T3 and T4. Dose-dependent inhibition of T4 synthesis was observed with 6-day exposures (Days 8–14) to three reference TDCs, methimazole (MMI), propylthiouracil (PTU), and sodium perchlorate (NaClO₄), yielding average IC₅₀ values of 0.50 (SE 0.01) μM, 0.96 (SE 0.18) μM, and 32.89 (SE 5.46) μM, respectively, while the negative control (methomyl) showed no effect. No cytotoxicity was observed for any of the reference compounds.
Conclusions:
In summary, the primary human thyrocyte 3D microtissue assay using ELISA represents a promising strategy for evaluating and prioritizing potential TDCs based on human hazard-related bioactivity. Moreover, the model enables the identification of compounds acting directly on the thyroid and supports simultaneous evaluation of multiple MIEs (e.g., TPO, NIS, TSHR) for small-molecule screening in both agriscience and therapeutic research.