Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

Microglia are brain-resident phagocytic cells, which originate from a population of myeloid progenitors from the yolk sac during embryonic development [16, 23, 35] and are maintained through self-renewal without influx of peripheral cells during adult life [1, 4]. Microglia are key players in central nervous system (CNS) homeostasis, fulfilling essential roles in neurodevelopment, adult synaptic plasticity, and brain immunity [32, 34]. In the adult brain, microglia act as surveyors of the local environment to sustain homeostasis and are therefore highly sensitive to changes associated with damage, inflammation, or infection within and outside the CNS. In order to interact with their environment, microglia exhibit a broad range of sensory mechanisms and specific cellular responses, the outcome of which can be both neuroprotective as well as a neurotoxic [22].

During the process of normal aging, the microglial phenotype appears to shift to a primed or more active-prone state [22, 30], the main reasoning behind microglia being linked to pathology in neurodegenerative disorders such as Alzheimer’s disease (AD) [21], Parkinson’s disease (PD) [33], and multiple sclerosis (MS) [24]. Their role as possible contributors to disease has been complemented by evidence for their involvement in the pathophysiology of developmental and psychiatric disorders, such as major depression disorder, bipolar disorder, schizophrenia, and autism [3, 7], either through modulation of neuroinflammation or neuronal plasticity. However, their role in disease pathology appears ambiguous since microglia also display beneficial and restorative functions [36].

Research on microglia function and their role in health and disease has mostly been carried out ex vivo using immunohistochemistry and in vivo using murine models. The isolation of microglia from the brains of various genetic mouse models has greatly facilitated our understanding of basic microglia characteristics in health and disease [9]. Nevertheless, these models are of limited value in relation to human CNS disorders. Studies into human microglia function have highlighted similarities but also crucial differences between mice and humans [38]. Added difficulty comes in the form of various CNS disorders for which animal models are not available or fail to reconstitute important human symptoms. Therefore, to investigate the role of microglia in human context it is crucial to study human primary microglia.

In order to specifically study multiple aspects of human microglia, obtaining pure microglia populations from post-mortem human brain samples is essential. To this aim, we have adapted the human microglia isolation method of Dick et al. [12], in turn based on a rat isolation protocol [37], for the use of post-mortem human brain tissue. This led to a procedure for the rapid isolation of pure human microglia based on cell density separation and capture of CD11b-positive cells using magnetic beads [25]. A major advantage of this isolation procedure in comparison with generally used microglia isolation methods [11] is the omission of effects due to culture and adherence in the procedure, as it allows for direct analysis of isolated microglia. Using this technique, we determined that based on membrane expression of CD45 and CD11b, microglia can be distinguished from autologous peripheral macrophages based on fluorescence intensity [25]. Furthermore, we demonstrated that microglia show a minimal response to lipopolysaccharide (LPS), indicating a tight regulation of inflammatory responses. Finally, we revealed differences in microglial size, granularity, and CD45/CD11b expression in white matter microglia from MS donors, when compared to non-MS donors [26], showing that microglial phenotype reflects neuropathological changes. Yet, to effectively study primary human microglia on a larger scale, there is an urgent need for thorough validation of available protocols and an understanding of the effects of clinical diagnosis and ante- and post-mortem variables on isolated microglia.

Since the development of our procedure for the isolation of human microglia in 2012 [25], we performed microglia isolations from over a hundred brain donors from the Netherlands Brain Bank. In addition to our previously published method, we have also developed a faster protocol that reduces the total isolation time, while maintaining similar or higher viable cell yield. Here we set out to validate the practical aspects of human post-mortem microglia isolations and describe the effects of clinical diagnosis and ante- and post-mortem variables on microglial purity and phenotype, such as post-mortem delay (PMD) and cerebrospinal fluid (CSF) pH, and discuss further application possibilities of isolated human microglia.