Alveolar macrophages (AMs) play critical roles in maintaining lung homeostasis and orchestrating the immune responses. Although the essential factors known for AM development have been identified, currently an optimal in vitro culture system that can be used for studying the development and functions of AMs is still lacking. In this study, we report the development of an optimized culture system for generating AM-like cells from adult mouse bone marrow and fetal liver cells on in vitro culture in the presence of a combination of GM-CSF, TGF-β, and peroxisome proliferator–activated receptor γ (PPAR-γ) agonist rosiglitazone. These AM-like cells expressed typical AM surface markers sialic acid–binding Ig-like lectin-F (Siglec-F), CD11c, and F4/80, and AM-specific genes, including carbonic anhydrase 4 (Car4), placenta-expressed transcript 1 (Plet1), eosinophil-associated RNase A family member 1 (Ear1), cell death–inducing DNA fragmentation factor A–like effector c (Cidec), and cytokeratin 19 (Krt19). Similar to primary AMs, the AM-like cells expressed alternative macrophage activation signature genes and self-renewal genes. Moreover, this culture system could be used for expansion of bronchoalveolar lavage fluid–derived AMs in vitro. The AM-like cells generated from bone marrow resembled the expanded bronchoalveolar lavage fluid–derived AMs in inflammatory responses and phagocytic activity. More importantly, these AM-like cells could be obtained in sufficient numbers that allowed genetic manipulation and functional analysis in vitro. Taken together, we provide a powerful tool for studying the biology of AMs.

肺泡巨噬细胞 (AMs) 在维持肺稳态和协调免疫反应方面发挥着关键作用。尽管已经确定了已知的 AM 发展的基本因素，但目前仍缺乏可用于研究 AMs 发展和功能的最佳体外培养系统。在这项研究中，我们报告了一种优化培养系统的开发，用于在 GM-CSF、TGF-β 和过氧化物酶体增殖剂的组合存在的情况下，通过体外培养从成年小鼠骨髓和胎肝细胞中产生 AM 样细胞-活化受体γ（PPAR-γ）激动剂罗格列酮。这些 AM 样细胞表达典型的 AM 表面标志物唾液酸结合 Ig 样凝集素-F (Siglec-F)、CD11c 和 F4/80，以及 AM 特异性基因，包括碳酸酐酶 4 ( Car4)、胎盘表达转录物 1 ( Plet1 )、嗜酸性粒细胞相关核糖核酸酶 A 家族成员 1 ( Ear1 )、细胞死亡诱导 DNA 片段化因子 A 样效应物 c ( Cidec ) 和细胞角蛋白 19 ( Krt19)）。与原代 AM 类似，AM 样细胞表达替代巨噬细胞激活特征基因和自我更新基因。此外，该培养系统可用于体外扩增支气管肺泡灌洗液衍生的 AM。从骨髓产生的 AM 样细胞在炎症反应和吞噬活性方面类似于扩大的支气管肺泡灌洗液来源的 AM。更重要的是，可以获得足够数量的这些 AM 样细胞，从而允许在体外进行基因操作和功能分析。总之，我们为研究 AM 的生物学提供了一个强大的工具。