The SENSE (Simple prEservatioN of Single cElls) method for cryopreservation enables the generation of single-cell immune profiles from whole blood.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA940678 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We developed and validated a simple method for viable cryopreservation of whole blood, without any preprocessing, Simple prEservatioN of Single cElls (SENSE), with granulocyte depletion for generating high-quality single-cell profiles. We performed rigorous in-depth characterization of the SENSE method on clinical blood samples and compared it to the conventional multistep density-gradient isolation of peripheral blood mononuclear cells (PBMCs) method. The SENSE method is an effective and simple solution for the cryopreservation of blood samples in clinics/labs and single cell profiles generation.
2023-11-28 | GSE226557 | GEO
Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.
2019-07-25 | GSE127248 | GEO
Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.
2019-07-25 | GSE127246 | GEO
Project description: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.
2023-07-05 | GSE234069 | GEO
Project description:With the growing interest in studying primary tissue samples by single cell transcriptome analysis, there is an emerging demand for a preservation strategy that enables sample transportation and storage. In this study, we describe a simple and general strategy that preserves primary tissues at hypothermic temperature. Using FACS and single-cell RNAseq, we demonstrated the effectiveness of this strategy in maintaining cell viability, cell population heterogeneity, and cell transcriptome integrity for primary tissues that underwent up to 3 days of preservation.
2018-02-21 | GSE88953 | GEO
Project description:As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating preservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservationrelated subtle micro-cellular damage needs further study
2020-10-03 | GSE158929 | GEO
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.
2024-03-26 | GSE252701 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE24753: Genome-wide analysis of the effect of cryopreservation on peripheral blood mononuclear cells GSE24755: Genome-wide analysis of the effect of long-term cryopreservation on peripheral blood mononuclear cells GSE24757: Genome-wide analysis of the effect of long-term freezing of PAXgene Blood RNA tubes Refer to individual Series
2011-07-21 | E-GEOD-24758 | biostudies-arrayexpress
Project description:IntroductionCurrent multistep methods utilized for preparing and cryopreserving single-cell suspensions from blood samples for single-cell RNA sequencing (scRNA-seq) are time-consuming, requiring trained personnel and special equipment, so limiting their clinical adoption. We developed a method, Simple prEservatioN of Single cElls (SENSE), for single-step cryopreservation of whole blood (WB) along with granulocyte depletion during single-cell assay, to generate high quality single-cell profiles (SCP).MethodsWB was cryopreserved using the SENSE method and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved using the traditional density-gradient method (PBMC method) from the same blood sample (n=6). The SCPs obtained from both methods were processed using a similar pipeline and quality control parameters. Further, entropy calculation, differential gene expression, and cellular communication analysis were performed to compare cell types and subtypes from both methods.ResultsHighly viable (86.3 ± 1.51%) single-cell suspensions (22,353 cells) were obtained from the six WB samples cryopreserved using the SENSE method. In-depth characterization of the scRNA-seq datasets from the samples processed with the SENSE method yielded high-quality profiles of lymphoid and myeloid cell types which were in concordance with the profiles obtained with classical multistep PBMC method processed samples. Additionally, the SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. Overall, the SENSE and PBMC methods processed samples exhibited transcriptional, and cellular communication network level similarities across cell types with no batch effect except in myeloid lineage cells.DiscussionComparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.
| S-EPMC10713715 | biostudies-literature
Project description:Simple oligonucleotide-based multiplexing of single-cell chromatin accessibility
| PRJNA683040 | ENA