DOI: 10.5061/dryad.j9kd51cjv
title: Technical evaluation and standardization of the human thyroid microtissue assay

description: The success and sustainability of U.S. EPA efforts to reduce, refine, and
 replace in vivo animal testing depends on the ability to translate
 toxicokinetic and toxicodynamic data from in vitro and in silico new
 approach methods (NAMs) to human-relevant exposures and health outcomes.
 Organotypic culture models employing primary human cells enable
 consideration of human health effects and inter-individual variability,
 but present significant challenges for test method standardization,
 transferability, and validation. Increasing confidence in the information
 provided by these in vitro NAMs requires setting appropriate performance
 standards and benchmarks, defined by the context of use, to consider human
 biology and mechanistic relevance without animal data. The human thyroid
 microtissue assay utilizes primary human thyrocytes to reproduce
 structural and functional features of the thyroid gland that enable
 testing for potential thyroid disrupting chemicals. As a variable-donor
 assay platform, conventional principles for assay performance
 standardization need to be balanced with the ability to predict a range of
 human responses. The objectives of this study were to 1) define the
 technical parameters for optimal donor procurement, primary thyrocyte
 qualification, and performance in the human thyroid microtissue assay, and
 2) set benchmark ranges for reference chemical responses. Thyrocytes
 derived from a cohort of 32 demographically-diverse euthyroid donors were
 characterized across a battery of endpoints to evaluate morphological and
 functional variability. Reference chemical responses were profiled to
 evaluate the range and chemical-specific variability of donor-dependent
 effects within the cohort. The data informed minimum acceptance criteria
 for donor qualification and set benchmark parameters for method transfer
 proficiency testing and validation of assay performance.
description: # Technical Evaluation and Standardization of the Human Thyroid
 Microtissue Assay **SUPPLEMENTARY TABLES LEGENDS**   **Supplementary Table
 1. Human Thyroid Microtissue (hTMT) Medium.** Formulation of hTMT medium
 components, vendor, catalog number, and final working concentrations.     
 **Supplementary Table 2. Assay Performance Parameters.** Performance
 criteria were established from the T4 median, median absolute deviation
 (MAD), and range values derived from the qualified donor cohort under
 assay-specific conditions with required priority levels for all metrics. 
   **Supplementary Table 3. Donor Procurement Parameters.** Donor
 procurement specifications extrapolated from linear regression modeling
 using the acceptance criteria defined for thyroglobulin, thyroxine (T4),
 and triiodothyronine (T3). Not applicable (NA).   **Supplementary Table 4.
 Multiple Linear Regression Statistics.** Multiple linear regression model
 statistics for microtissue biomass, thyroglobulin, T4, T3, and T4/T3
 ratio. Analysis of Variance (ANOVA) parameters include the residual sum of
 squares (SS), degrees of freedom (DF), mean square (MS), F statistic, and
 p-value. The parameter estimates include the dependent variable, estimate,
 standard error, 95% confidence interval (CI), t statistic, p-value, and
 p-value summary. The goodness-of-fit reports the degrees of freedom (DF),
 multiple R value, R<sup>2</sup> value, adjusted
 R<sup>2</sup> value, sum of squares, Sy.x, root mean squared
 error (RMSE), and corrected Akaike Information Criterion (AICc). The
 multicollinearity reports the independent variables, the variance
 inflation factor (VIF), and R<sup>2</sup> with other
 variables. The normality of the residuals statistics and data summary are
 also reported.    **Supplementary Table 5. Simple Linear Regression
 Statistics.** Simple linear regression model statistics for microtissue
 thyroglobulin, T4, and T3 relative to age and BMI. Best fit values, 95%
 confidence intervals, model goodness-of-fit, significance of slope, model
 equation, and data summary are reported. Dfn and DFd are the degrees of
 freedom for the numerator and denominator of the F ratio, respectively.  
 **Supplementary Table 6. Literature-derived Reference Chemical Potency
 Values.** Specifications for chemical name, target MIE, species, assay
 method, assay source material, assay type, potency, potency 95% confidence
 interval (CI), potency unit, potency category, and references are noted.
 n/a: not applicable   **SUPPLEMENTARY FIGURE LEGENDS**   **Supplementary
 Figure 1. Donor Micrographs at Day 14.** Representative micrographs across
 three treatment groups (Tx-1, Tx-2, Tx-3) for each donor evaluated in the
 study (N=32). Scale bar is 1,000 µM.   **Supplementary Figure 2. Endpoint
 Correlations.** Pearson correlation of characterization endpoints at day
 14 in the (A) Tx-1 and (B) Tx-3 treatment groups. Values greater than 0
 indicate a positive correlation, less than 0 a negative correlation, and 0
 as no correlation.   **Supplementary Figure 3. Evaluation of Thyroid
 Hormone Synthesis Inhibition by Reference Chemical – Methomyl.** (A)
 Population-level concentration-response modeling of the total (N=32)
 versus qualified (N=24) donor cohort. The values were normalized by donor
 and plotted in aggregate for data modeling. The red band represents the
 95% confidence interval of the model fit. The gray band indicates the 95%
 prediction interval where 95% of future donors would be expected to
 exhibit bioactivity. **Supplementary Figure 4. Reference Chemical
 Cytotoxicity Plots.** Population-level concentration-response modeling of
 ATP measurements at day 14 for the total (N=32) versus qualified (N=24)
 donor cohort in response to (A) methomyl, (B) methimazole, (C)
 6-propyl-2-thiouracil, and (D) sodium perchlorate. The values were
 normalized by donor and plotted in aggregate for data modeling. The red
 band represents the 95% confidence interval of the model fit. The gray
 band indicates the 95% prediction interval where 95% of future donors
 would be expected to exhibit bioactivity. The horizontal dashed line
 represents the baseline. **Supplementary Figure 5. Assay Plate Maps.** The
 96-well plate map defined by the assay workflow on designated days for
 seeding (Day 0), medium exchanges (Day 2-8), and chemical treatments (Day
 8-14). Control wells (Tx-1, Tx-2, Tx-3) and the chemical concentrations
 (µM) are noted. The table specifies TSH supplementation, compound
 treatments, concentration range and units, technical replicates, the
 molecular initiating event (MIE) target of the treatment, and chemical
 classification as defined by the assay context. Not applicable (NA).