DMSO cryopreservation is the method of choice to preserve cells for droplet-based single-cell RNA sequencing
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Combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has recently become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomic 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.
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Ambient RNA contamination with highly abundant transcripts suggests cell damage by methanol fixation.
“we observed ambient RNA contamination with highly abundant transcripts suggesting cell damage by methanol fixation [22].”
From Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows (2020) · cites this paper as [22]
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- Authors
- Wohnhaas, C. T., Leparc, G. G., Fernandez-Albert, F., Kind, D., Gantner, F., Viollet, C., Hildebrandt, T., & Baum, P.
- Affiliations
- Boehringer Ingelheim (Germany) (DE)
- Corresponding author
- Baum, Patrick
- Venue
- Scientific Reports
- Published
- 2019-07-23
- Volume
- 9
- Issue
- 1
- Pages
- 10699-10699
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC6650608
- OpenAlex
- W2964240868
- Cited by
- 105
- References
- 31
- Citations by year
- 2026: 5 · 2025: 14 · 2024: 10 · 2023: 17 · 2022: 23 · 2021: 21 · 2020: 15
- FWCI
- 4.97
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- KOPS (University of Konstanz) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- Universität Konstanz
- National Institutes of Health (HEK293)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Physical Sciences › Engineering › Biomedical Engineering › Innovative Microfluidic and Catalytic Techniques Innovation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- cell cryopreservation, dimethyl sulfoxide, cell preservation, methanol fixation, gene expression correlation, cell integrity, monocyte-derived macrophages, immune cells, cell lines
- MeSH terms
- Dimethyl Sulfoxide, Humans, Cryopreservation, Sequence Analysis, RNA, Gene Expression Profiling, Single-Cell Analysis
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