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

Abstract

Summary Microglia play a key role in brain development, normal homeostasis, and neurodegenerative disorders. Single-cell technologies have led to important findings about microglia, with many animal model studies using single-cell RNA sequencing (scRNA-seq), whereas most human specimen studies using archived frozen brains for single-nucleus RNA sequencing (snRNA-seq). However, microglia compose a small proportion of the total brain tissue; snRNAseq depletes expression of microglia activation genes that characterize many diseases. Here we examine the use of purified, cryopreserved microglia for scRNA-seq. Comparison of scRNA-seq on paired fresh and cryopreserved microglia from rhesus monkeys revealed a high level of correlation of gene expression between the two conditions. Disease-related genes were relatively unaffected, but an increase in immediate-early gene expression was present in cryopreserved cells. Regardless, changes in immediate-early gene expression are still detectable. Cryopreservation of microglia is a suitable procedure for prospectively archiving samples.

Morphine Suppresses Peripheral Responses and Transforms Brain Myeloid Gene Expression to Favor Neuropathogenesis in SIV Infection

“…At necropsy, blood-borne cells were cleared by perfusion with PBS containing 1U/mL heparin. Microglia/macrophage-enriched cellular isolation from the brain was performed using our previously described procedure of physical and enzymatic disassociation followed by Ficoll gradient purification (10, 137), and then cryopreserved in the presence of 10% DMSO / 90% fetal bovine serum and stored over liquid nitrogen. Brain regions were dissected by a board-certified pathologist, frozen, and stored at −80° C.…”

“…scRNA-seq procedures were completed using our previously described methods (137, 142). Briefly, cryopreserved microglia were treated with 1% DNase (Sigma Aldrich), washed, stained with UV-blue live/dead assay (Invitrogen, Waltham, MA).…”

“…For the caudate those same substitutions were made, in addition morphine 79T (CI83) was substituted for 75T in the morphine group. scRNA-seq procedures were completed using our previously described methods (137,142). Briefly, cryopreserved microglia were treated with 1% DNase (Sigma Aldrich), washed, stained with UV-blue live/dead assay (Invitrogen, Waltham, MA).…”

Stress relief: emerging methods to mitigate dissociation-induced artefacts

“…Several studies have demonstrated that exposing mammalian cells to sub-physiological temperatures invokes a coordinated cellular response involving induction of cold-shock proteins and modulation of translation, metabolism, cell cycle and the cell cytoskeleton [39]. Therefore, although cold digestion prevents, to a large extent, the dissociation-induced artefacts, it is still possible to alter in some way the in vivo molecular profile of the cells, for example, cryopreserved cells experiment some induction of earlyresponse genes [40]. Of note, cells isolated using standard procedures also commonly experience cold-stress during several steps of the protocol, such as centrifugation and transfer of cells, as these steps are performed at 4°C and on ice, respectively.…”

Cell-associated Transcriptional Alterations in the Retinal of Alzheimer’s Disease

“…While total transcript can be extracted from archival tissue using single-nuclei RNA-seq, detecting activation of state-associated genes is inconsistent between brain studies. On one hand, disease-activated genes were preserved in frozen microglial nuclei 24,25 . However, a meta-analysis of publicly available microglial datasets indicates that microglial activation genes may be depleted in single-nuclei RNA-seq datasets, including genes involved in AD 26 .…”