功能性和自我耐受性 T 细胞库的生成是一个复杂的过程，依赖于胸腺微环境，并且主要依赖于其细胞外基质 (ECM) 的特性。胸腺上皮细胞 (TEC) 在胸腺生成、通过过滤自身反应克隆来培育和选择发育中的 T 细胞方面至关重要。经验证明 TEC 对 ECM 提供的物理和化学线索特别敏感，经典的单层细胞培养会导致功能快速丧失直至死亡。由于这种微妙的维护加上相对稀有，尽管在体外模拟胸腺生物学具有很高的风险，但能够忠实地大规模模仿 TEC 生态位并随着时间的推移的模型仍然缺乏。在这里，我们描述了多细胞人类胸腺类器官模型的形成，其中TEC隔室源自人类诱导多能干细胞（iPSC），并与原代早期胸腺祖细胞在三维（3D）基于纤维蛋白的水凝胶中重新聚集。该模型满足了当前对可扩展培养系统的需求，该系统可在体外复制胸腺微环境并展示其功能，即产生 T 细胞和支持胸腺类器官在数周内生长的能力。因此，我们提出了一种通过 iPSC 衍生的类器官实现胸腺功能的实用体外模型，这将有利于 TEC 生物学和离体 T 细胞生成的研究。

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.