类器官 3D 系统是研究发育和疾病的强大平台。最近，源自成年小鼠和人类干细胞的肺类器官模型的细胞组成和结构复杂性显着增加。然而，具有清晰的整合内皮室的小鼠肺类器官系统仍然缺失。在这里，我们描述了一种新方法，通过将 FACS 分选的肺内皮细胞 (EC) 显微注射到分化的类器官培养物中，为我们发表的支气管肺泡肺类器官 (BALO) 模型增加了另一个层次的复杂性。在显微注射之前，从幼年小鼠的肺匀浆（LH）中获得的EC表达典型的EC标记物，例如CD31和血管内皮（VE）-钙粘蛋白，并显示出管形成能力。显微注射后，EC 围绕 BALO 的肺泡样区室，与 I 型 (AECI) 和 II 型 (AECII) 肺泡上皮细胞对齐，如共聚焦和电子显微镜所示。值得注意的是，还检测到了 Car4 和 Aplnr 的表达，表明培养的 EC 中存在 EC 微血管表型。此外，在脂多糖（LPS）和甲型流感病毒（IV）损伤上皮细胞后，内皮化BALO（eBALO）释放促炎细胞因子，导致EC中细胞间粘附分子1（ICAM-1）上调。总之，我们首次描述了将 EC 纳入肺类器官肺泡结构的类器官模型，不仅增加了我们之前模型的细胞和结构复杂性，而且还为模型肺内皮对离体损伤的反应提供了合适的利基。

Organoid 3D systems are powerful platforms to study development and disease. Recently, the complexity of lung organoid models derived from adult mouse and human stem cells has increased substantially in terms of cellular composition and structural complexity. However, a murine lung organoid system with a clear integrated endothelial compartment is still missing. Here, we describe a novel method that adds another level of intricacy to our published bronchioalveolar lung organoid (BALO) model by microinjection of FACS-sorted lung endothelial cells (ECs) into differentiated organoid cultures. Before microinjection, ECs obtained from the lung homogenate (LH) of young mice expressed typical ECs markers such as CD31 and vascular endothelial (VE)-Cadherin and showed tube formation capacity. Following microinjection, ECs surrounded BALO´s alveolar-like compartment aligning with both alveolar epithelial cells type I (AECI) and type II (AECII), as demonstrated by confocal and electron microscopy. Notably, expression of Car4 and Aplnr was as well detected, suggesting presence of EC microvascular phenotypes in the cultured ECs. Moreover, upon epithelial cell injury by lipopolysaccharides (LPS) and influenza A virus (IV), endothelialized BALO (eBALO) released proinflammatory cytokines leading to the upregulation of the intercellular adhesion molecule 1 (ICAM-1) in ECs. In summary, we characterized for the first time a organoid model that incorporates ECs into the alveolar structures of lung organoids, not only increasing our previous model ́s cellular and structural complexity but also providing a suitable niche to model lung endothelium responses to injury ex vivo.