Establishment of a microwell-array-based miniaturized thymic organoid model suitable for high-throughput applications

T cell development depends critically on the thymic stroma—in particular, the diverse array of functionally distinct thymic epithelial cell (TEC) types. However, a robust in vitro thymus model mimicking the native thymus and compatible with medium-/high-throughput analyses is currently lacking. Here, we demonstrate a high-density microwell-array-based miniaturized thymus organoid (mTO) model that supports T cell commitment and development, possesses key organizational characteristics of the native thymus, and is compatible with live imaging and medium-/high-throughput applications. We establish the minimum cellular input required for a functional mTO and show that mTO TEC phenotype and complexity closely mirror those of the native thymus. Finally, we use an mTO to probe the role of fetal thymic mesenchyme, revealing a requirement beyond maintenance of Foxn1 in differentiation/maintenance of mature TEC sub-populations. Collectively, mTOs present an in vitro model of the native thymus adaptable to medium-/high-throughput applications and validated for exploration of thymus and thymus organoid biology.