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Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns

ABSTRACT: We report the effect of cryopreservation on isolated microglia and macrophages from the brains of rhesus monkeys. At the time of necropsy, cells were isolated from the brain and either used fresh or cryopreserved. For single cell sequencing, isolates were purified for CD11b expression and captured and processed through the 10xGenomics platform following by sequencing. We find that cryopreservation altered the expression of approximately 2% of the genes. These were not enriched for any known microglia/macrophage set of differentially regulated genes, but was enriched in immediate early genes and the acute phase pathway. Assessment of the fresh and cryopreseved cells in comparison to a prior dataset from animals with encephalitis revelaed equal ability to detect differentially expresed genes. Thus, given the practical difficulty of freshly processing microglia from human and non-human primate brains for isolation and scRNAseq processing the same day, and the ability to archive sampoles for potential use in scRNAseq experiments, cryopreservation is an optimal choice for future analysis of these important brainn cells, and likely other cell types.

ORGANISM(S): Macaca mulatta

PROVIDER: GSE162663 | GEO | 2021/01/22

REPOSITORIES: GEO

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Project description:Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approaches to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site to- site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh (Samples labeled as FRE) or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO, Samples labeled as FBS), FBS/DMSO after a DNase step (a step often included in epigenetic studies, samples labeled as DNA), and cryopreservation using commercially available Recovery©media (Samples labeled as REC). In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further, demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

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Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns

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