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

Abstract

Single-cell transcriptomics allows characterization of cerebrospinal fluid (CSF) cells at an unprecedented level. Here, we report a robust cryopreservation protocol adapted for the characterization of fragile CSF cells by single-cell RNA sequencing (RNA-seq) in moderate- to large-scale studies. Fresh CSF was collected from twenty-one participants at two independent sites. Each CSF sample was split into two fractions: one was processed fresh, while the second was cryopreserved for months and profiled after thawing. B and T cell receptor sequencing was also performed. Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. We find no significant difference in cell type proportions and cellular transcriptomes between fresh and cryopreserved cells. Results were comparable at both sites and with different single-cell sequencing chemistries. Cryopreservation did not affect recovery of T and B cell clonotype diversity. Our CSF cell cryopreservation protocol provides an important alternative to fresh processing of fragile CSF cells.

Keywords: BCR sequencing; TCR sequencing; cerebrospinal fluid; cryopreservation; immune cells; single-cell RNA-seq.

Graphical abstract

Figure 1

Schematic representation of the study design CSF was collected via lumbar puncture and rapidly transported on ice for CSF cell processing. CSF cells were split into two fractions for fresh analysis or cryopreservation. CSF cells were analyzed using scRNA-seq.

Figure 2

Efficient CSF cell cryopreservation validated by 3′ single-cell transcriptomics (A) Post-quality control (QC) CSF cell counts in the fresh and cryopreserved (Cryo) sample pairs (p = 0.24). (B) Uniform manifold approximation and projection (UMAP) plot of 21 clusters/cell states color coded by their annotations. (C) UMAP indicating good representation of fresh and Cryo cells in each cluster. (D) Annotation of clusters using selected marker genes. Heatmap colors correspond to the proportion of cells in each cluster expressing cluster marker gene. (E) Bar chart indicating similar cell-type proportions in thirteen fresh and Cryo sample pairs. (F and G) Significant positive correlation (F) between the different cluster proportions from thirteen fresh and Cryo sample pairs (p = 0.93) and (G) between two fresh-fresh sample pairs (p = 0.98, 0.99). Each point represents a cluster color coded by its sample ID. See also Tables S1, S2, and S3.

Figure 3

Efficient CSF-cell cryopreservation validated by 5′ single-cell transcriptomics (A) Post- quality control (QC) CSF cell counts in the fresh and cryopreserved (Cryo) sample pairs (p = 0.37). (B) Uniform Manifold Approximation and Projection (UMAP) plot of 21 clusters color coded by their annotations. (C) UMAP indicating good representation of fresh and Cryo cells in each cluster. (D) Annotations of clusters using selected marker genes. Heatmap colors correspond to the proportion of cells in each cluster expressing cluster marker gene. (E) Bar chart indicating similar CSF cell type proportions in eight fresh-Cryo sample pairs. (F) Significant positive correlation between the different cluster proportions in eight fresh-Cryo sample pairs (p = 0.97). (G) Clonal rank indicating similar distribution of the T and B cell clonotypes in the eight fresh and Cryo CSF samples. See also Tables S1, S2, S3, and S4.