School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
Innovation Platform of Marine Drug Screening and Evaluation, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Front Immunol. 2022 Jul 6;13:920232. doi: 10.3389/fimmu.2022.920232. eCollection 2022.
The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity of harvested macrophages. Several previous studies assessed the detachment efficacies of enzymatic and non-enzymatic methods for harvesting the single cell suspensions of macrophages, but a comprehensive study assessing different dissociation methods and culture conditions for detaching functionally different macrophage populations has not yet been reported. In this study, the well-established GM-CSF and M-CSF differentiated bone marrow derived macrophage models (GM-BMDMs and M-BMDMs), we compared four commonly used enzymatic (trypsin and accutase) and non-enzymatic (PBS and EDTA) dissociation methods along with necessary mechanical detaching steps (scraping and pipetting) to evaluate the viable cell recovery and cell surface marker integrality of GM-BMDMs and M-BMDMs cultured on standard cell culture dish (TC dish), or on culture dish (noTC dish) that was not conditioned to enhance adherence. The data showed that accutase yielded a better recovery of viable cells comparing with PBS and EDTA, especially for tightly adherent GM-BMDMs on TC dishes, with a relatively higher level of detected cell membrane marker F4/80 than trypsin. An additional gradient centrifugation-based dead cell removal approach could increase the proportion of viable cells for TC cultured GM-BMDMs after accutase dissociation. Furthermore, transcriptome analysis was performed to evaluate the putative influence of culture dishes. At steady state, BMDMs cultured on noTC dishes exhibited more proinflammatory gene expression signatures (e.g. IL6, CXCL2 and ILlβ) and functions (e.g. TNF and IL17 signaling pathways). Similar inflammatory responses were observed upon LPS challenge regardless of culture conditions and differentiation factors. However, in LPS treated samples, the difference of gene expression patterns, signaling pathways and molecular functions between TC and noTC cultured BMDMs were largely dependent on the types of growth factors (M-CSF and GM-CSF). This observation might provide valuable information for macrophage studies.
培养皿表面的性质和用于分离贴壁细胞的技术很可能会影响收获的巨噬细胞的细胞活力和细胞膜蛋白完整性。先前有几项研究评估了酶和非酶方法用于收获巨噬细胞单细胞悬液的分离效果，但尚未有研究全面评估用于分离功能不同的巨噬细胞群体的不同解离方法和培养条件。在这项研究中，我们使用了经过充分验证的 GM-CSF 和 M-CSF 分化的骨髓来源的巨噬细胞模型（GM-BMDMs 和 M-BMDMs），比较了四种常用的酶（胰蛋白酶和 accutase）和非酶（PBS 和 EDTA）解离方法以及必要的机械分离步骤（刮擦和移液），以评估在标准细胞培养皿（TC 皿）或未进行增强附着处理的培养皿（noTC 皿）上培养的 GM-BMDMs 和 M-BMDMs 的活细胞回收率和细胞表面标志物完整性。数据显示，与 PBS 和 EDTA 相比，accutase 可以更好地回收活细胞，特别是对于 TC 培养皿上紧密附着的 GM-BMDMs，其检测到的细胞膜标志物 F4/80 水平相对较高，比胰蛋白酶高。在 accutase 解离后，采用基于梯度离心的死细胞去除方法可以增加 TC 培养 GM-BMDMs 中的活细胞比例。此外，还进行了转录组分析以评估培养皿的可能影响。在稳态下，在 noTC 培养皿上培养的 BMDMs 表现出更高的促炎基因表达特征（例如，IL6、CXCL2 和 ILlβ）和功能（例如，TNF 和 IL17 信号通路）。无论培养条件和分化因子如何，在 LPS 刺激后均观察到类似的炎症反应。然而，在 LPS 处理的样本中，TC 和 noTC 培养的 BMDMs 之间的基因表达模式、信号通路和分子功能的差异在很大程度上取决于生长因子的类型（M-CSF 和 GM-CSF）。这一观察结果可能为巨噬细胞研究提供有价值的信息。
