Table_1_Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation.XLSX

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.

小胶质细胞是中枢神经系统（CNS）的固有免疫细胞，它们在多种神经系统疾病的神经炎症发病机制中已被涉入。它们在神经炎症中的多种情境依赖性贡献才刚刚开始被阐明，这部分原因可归因于在体内研究小胶质细胞的挑战，以及缺乏易于操作的培养系统来研究小胶质细胞的功能。组织型脑切片培养提供了与组织相关的环境，使得对中枢神经系统固有细胞的研究成为可能，而对脑切片小胶质细胞表型的分析已提供了重要的见解，特别是在神经保护功能方面。在本研究中，我们利用RNA测序、nCounter®技术的直接数字基因表达定量以及针对单个小胶质细胞特征基因的靶向分析，对脑切片小胶质细胞相对于急性分离的同源细胞和二维（2D）原代小胶质细胞培养（一种广泛使用的体外替代品）进行了表征。使用单细胞和基于群体的方法分析发现，与体外细胞相比，脑切片小胶质细胞在保留经典小胶质细胞标记和整体基因表达方面表现出更强的保真度，更接近急性分离的成年小胶质细胞。我们表征了脑切片小胶质细胞在培养过程中的动态表型变化。与切片制备相关的机械损伤引发了一个初始的炎症期，但随着时间的推移而得到缓解。基于流式细胞术和基因表达谱分析，我们确定了两周的时间点为研究小胶质细胞对外源性刺激反应的最佳时间点，例如在培养介质中加入LPS、TNF和GM-CSF后观察到的诱导神经炎症变化。总之，这些发现表明，脑切片培养作为一种实验系统，在研究小胶质细胞功能及其生理学上，相较于体外培养小胶质细胞作为替代品具有优越性。