Preservation of cerebrospinal fluid cells to investigate intrathecal immunity in neurodegeneration


 Background: Cerebrospinal fluid (CSF) provides basic mechanical and immunological protection to the brain. Historically, analysis of CSF has focused on protein changes, yet recent studies of neurodegenerative diseases have shed light on cellular alterations. Due to low cell numbers in the CSF, only recently have advances in molecular biology techniques allowed for the investigation of disease-related cellular changes. Chief among these advances is single cell RNA sequencing (scRNAseq). However, concerns such as batch effects and time constraints call for a standardized method for long-term preservation of CSF immune cells. Results: We present a method for long-term cryopreservation of CSF immune cells for downstream scRNAseq analysis. Cells can be analyzed up to two years following CSF collection. On average, 4,961 live cells can be recovered from 10 mL CSF after cryopreservation, with no association between length of storage and cell viability. We demonstrate that scRNAseq detects CD8 + and CD4 + T cells, natural killer cells, plasma cells, B cells, and innate immune cells (monocytes and dendritic cells) within the CSF of 24 subjects. Conclusions: The ability to store CSF cells long-term will enable researches actively collecting CSF to investigate intrathecal immunity. This method will aid in the understanding of cellular and molecular changes to CSF immunity in neurodegenerative diseases.


BACKGROUND
The cerebrospinal fluid (CSF) provides insight into brain physiology of living individuals. Biochemical analysis of CSF is routinely utilized as a diagnostic tool in neurodegeneration (1,2). For example, in Alzheimer's disease (AD), changes in protein levels of total tau, phosphorylated tau and amyloid-β are indicative of disease pathology. However, while CSF biomarkers guide diagnosis of patients with neurodegenerative diseases, the cells patrolling the interstitial fluid are often centrifuged and discarded. Only in cases of extreme central nervous system inflammation, such as meningitis or encephalitis, are CSF cells utilized as a diagnostic. Blood cells, on the other hand, are routinely used to assess health and disease. The composition of blood and CSF cells are distinct (3), with CD4 + and CD8 + T cells, plasmacytoid dendritic cells, and CD56 high natural killer (NK) cells enriched in the CSF, while monocytes, granulocytes, myeloid dendritic cells, basophils, and B cells are not as abundant (1). CSF cell density is also drastically lower with 1,000-3000 cells per mL versus millions per mL for blood (3)(4)(5). In comparison to blood, T lymphocytes are the most abundant cell type of the CSF and are comprised of more activated, antigen experienced cells.
Most of the aforementioned studies have relied on freshy isolated CSF cells (3,5,6,(9)(10)(11)(12)(13)(14)(16)(17)(18)(19)(20), or merely extraction of genomic DNA or RNA from frozen cells (7,8). Analyzing fresh CSF cells provides the highest number of viable cells and the closest approximation of their endogenous physiology, but introduces batch effects and time restrictions. Conversely, cryopreservation allows for parallel analysis of multiple samples acquired longitudinally. Increased evidence for cellular changes within the CSF warrants a standardized protocol for the long-term preservation of CSF immune cells. Here we report a method for the long-term storage and subsequent analysis of CSF cells by scRNAseq. This method will enable researchers to interpret undiscovered neurodegenerative disease mechanisms.

RESULTS
We developed a standardized workflow for the isolation, cryopreservation and sequencing of CSF immune cells (Figure 1a). We first enumerated freshly isolated cells from 10 mL of CSF obtained by lumbar puncture from 18 subjects. Following centrifugation, we recovered an average of 15,121 (±2,948 s.e.m.) live cells per subject (Figure 1b). Prior to cryopreservation, cells had an average viability of 58% (±6.8% s.e.m.; Figure 1c). After cryopreservation, cells were thawed at 37 • C and live cells were sorted using a live/dead dye (Figure 1d). After thawing, an average of 4,961 (± 506.1 s.e.m.) live cells were recovered by flow cytometry sorting ( Figure  1e) and 71% (± 2.0% s.e.m.) of scatter-gated cells were viable (Figure 1f). While a considerable number of cells are lost with thawing, we did not detect an impact of the length of storage and cell viability (R 2 =0.0079; Figure  1g Figure 2c). Importantly, we did not detect platelet genes, such as PPBP, confirming that our CSF samples were not contaminated with blood.

DISCUSSION
Most studies performed on human cells rely on peripheral blood mononuclear cells. However, utilizing peripheral cells as a read-out of immune changes in neurodegeneration limits our ability to understand central immunity. CSF cells, on the other hand, provide a way to directly study immunology in the central nervous system. Indeed, recent studies of intrathecal immunity by us and others have shown this understudied immune compartment to be relevant to the pathobiology of neurodegenerative diseases (3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Yet CSF cells have been widely understudied because of the invasive method of CSF extraction (typically lumbar puncture), and because cells are often discarded for proteomic analysis. Cells not discarded are typically analyzed fresh-typically by flow cytometry-which limits the scope of analysis.
Our simple and quick method allows for the preservation of an average 4,961 live cells after thawing, with no relationship between length of storage and cell viability. Importantly, the composition of cells from our method resembles that of freshly processed CSF cells reported in the literature. We find 66% of intrathecal cells are CD4 + T cells and 19% are CD8 + T cells, which agrees with the 60-83% and 11-20% range reported by others, respectively (3,21). We find that monocytes and dendritic cells combined make up 8.5% of CSF cells, which is similar to a previous study that found that monocytes make up 5-12% and dendritic cells comprise less than 4% of intrathecal cells (3). Unfortunately, we were not able to determine the proportion of monocytes and dendritic cells separately, as they did not form distinct clusters in our sequencing analysis. We also detected small proportions of NK cells (1.8%), plasma cells (0.49%) and B cells (0.31%). Han et al. report that natural killer cells make up 5%-which is slightly higher than our observations-and others report that B cells typically make up less than 1% of CSF cells (3,21). Altogether, while we lose minor proportions of NK and B cells, the composition of cells processed using our method of long-term preservation largely reflects that of freshly isolated CSF cells.
While studying fresh CSF cells provides maximal cell recovery, our method preserves major CSF cell types and their endogenous composition. Importantly, cryopreservation eliminates unavoidable batch effects and time constraints introduced from analyzing freshly isolated CSF cells. Therefore, cryopreservation of CSF cells allows for more complex and meaningful experiments on samples acquired longitudinally.

CONCLUSIONS
In conclusion, we provide a detailed method for long-term preservation of CSF cells and subsequent analysis by scRNAseq. We demonstrate that researchers can preserve CSF cells long-term and obtain valuable molecular information. However, we caution that freezing and thawing under this protocol may select for cells that better survive this process. Nonetheless, we anticipate that this simple protocol will enable other researchers to study CSF cells in a more robust and standardized way, providing important insight into intrathecal immunity in neurodegenerative diseases.

Study participants
Samples were acquired through the Stanford Brain Rejuvenation Program, the NIA funded Stanford Alzheimer's Disease Research Center (ADRC), the University of California at San Francisco ADRC and the University of California at San Diego ADRC. Collection of CSF was approved by the Institutional Review Board of each university; written consent was obtained from all subjects. A total of 34 living subjects were used in this study. The 34 subjects included 10 subjects with Parkinson's disease (PD), 3 with Lewey-body dementia (LBD), 4 subjects with Alzheimer's disease (AD), 5 patients with Mild Cognitive Impairment (MCI), and 12 healthy controls.

Cryopreservation of CSF cells
CSF was collected by lumbar puncture, then centrifuged at 300 rcf for 10 minutes at 4 • C to pellet immune cells. Importantly, CSF samples were checked for blood contamination by examining the pellet for the presence of red blood cells by eye. An example of a CSF sample contaminated with blood is shown in Supplemental Figure 1a. Note that cells should remain at 4 • C until they are further processed, but it is best to freeze the cells as quickly as possible to limit cell death. The supernatant (cell free CSF) was aliquoted, carefully leaving behind 100 μl of CSF with the pelleted cells. 100 μl of CSF was left so that cells were concentrated enough for counting and viability measurements. The pelleted cells were then gently resuspended in the 100 μl CSF and 10 μl of resuspended cells were then removed for counting. Importantly, cells were gently resuspended by first tapping the bottom of the tube and then gently triturating 10 times, making sure not to touch the pipette tip to the edge of the tube. Then 10 μl CSF was removed and mixed with 10 μl trypan blue to assess red blood cell content and viability. Cells were then visualized on a TC20 automated cell counter (BioRad) and cell number, viability and the presence or absence of red blood cells was recorded. An example of a CSF sample contaminated with blood is shown in Supplemental Figure 1b. CSF samples contaminated with blood were discarded. The resuspended cells were then mixed with 900 μl Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific). This medium is an optimized version of the typical freezing medium, containing high-glucose Dulbecco's Modified Eagle Medium with 10% serum and 10% dimethyl sulfoxide. We utilized this medium because it is quality tested for pH, osmolality, sterility, and endotoxin and each lot is quality tested on CHO-K1 cells. The freezing medium was first thawed at 37 • C, aliquoted, and stored at -20 • C. Before use, the medium was thawed at 37 • C and kept on ice. After each aliquot was thawed, the freezing medium was stored at 4 • C for up to one month. All samples were frozen overnight at −80 • C in a Mr. Frosty freezing container (Thermo Fisher Scientific) and transferred the following day to liquid nitrogen for storage. CSF cells were stored in liquid nitrogen on average 266 days.