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A structured evaluation of cryopreservation in generating single cell transcriptomic from cerebrospinal fluid

ABSTRACT: Recent advances in single-cell transcriptomics now allow characterization of CSF cells at an unprecedented scale and precision. With the availability of different single cell technologies with which to characterize CSF cells, there is a need for a simple yet robust protocol to cryopreserve CSF cells. In this study, we report a reliable cryopreservation protocol adapted for CSF cells that facilitates the characterization of these rare, fragile cells in moderate to large scale studies. To establish this protocol, excess CSF was collected following diagnostic lumbar punctures in twenty-one patients at two independent sites, and CSF cells were isolated. Each cell sample was split into two fractions for single cell analysis using one of two possible chemistries: 3’ sc-RNA-Sequencing or 5’sc-RNA-Sequencing. One cell fraction was processed fresh while the second sample was cryopreserved and profiled at a later time after thawing. B and T cell receptor sequencing were performed in a subset of samples. Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. The proportion of all cell types was similar between the fresh and the cryopreserved cells processed, and cellular transcriptomes were not significantly different. Results were comparable at both performance sites and with different single cell sequencing chemistries. Cryopreservation also did not affect recovery of T and B cell clonotype diversity. Hence, our cryopreservation protocol for CSF cells provides an important alternative to fresh processing of fragile CSF cells which is also resource-intensive: cryopreservation enables the involvement of research sites with limited capacity for experimental manipulation and reduces technical variation by enabling batch processing and pooling of samples. sequencing (scRNA-seq) to analyze the diversity of GCs in the intestine.

ORGANISM(S): Homo sapiens

PROVIDER: GSE234069 | GEO | 2023/07/05

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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A structured evaluation of cryopreservation in generating single cell transcriptomic from cerebrospinal fluid

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