Technical evaluation and standardization of the human thyroid microtissue assay

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.