Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, United States of America.
PLoS One. 2021 Apr 9;16(4):e0249117. doi: 10.1371/journal.pone.0249117. eCollection 2021.
Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7-9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.
巨噬细胞和树突状细胞 (DCs) 是先天免疫细胞，在抵御病原体方面发挥着关键作用。骨髓来源的巨噬细胞 (BMDM) 和树突状细胞 (BMDC) 的体外培养是免疫研究的重要方法。通常，使用市售的重组 GM-CSF 来生成 BMDCs，也用于培养肺泡巨噬细胞。我们已经生成了一种新的表达鼠 GM-CSF 的 HEK-293T 细胞系，该细胞系可将 GM-CSF 分泌到完全培养基中，其产量很高（约 180ng/ml），可替代市售 GM-CSF。使用 GM-CSF HEK293T 细胞系（称为 supGM-CSF）对树突状细胞的分化和各种标志物的表达进行了动力学评估，并与纯化的商业 GMCSF 进行了直接比较。细胞培养 7-9 天后，与商业纯化 GM-CSF 相比，supGM-CSF 产生的活细胞数量增加了一倍。除了分化 BMDCs 之外，supGM-CSF 还可用于培养功能活跃的肺泡巨噬细胞。总之，我们的结果表明，我们的 GM-CSF HEK293T 细胞系的上清液支持小鼠 BMDC 或肺泡巨噬细胞的培养，为纯化 GM-CSF 提供了一种经济的替代方法。
