Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations
Open accessPreprint — published as 10.3390/ijms21217944 · Paper page
Abstract
Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37°C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4°C. These findings emphasize the risk of introducing technical biases and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.
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A recent study proposed a modified mechanical method to isolate all CNS cell types and appeared to increase neuronal yields, but lacked biological replicates for proper analysis of cell-type abundance and reproducibility.
“One recent study proposed a modified mechanical method to isolate all CNS cell types and appeared to succeed in significantly increasing neuronal cell yields during dissociation. However, the study lacked biological replicates needed to perform proper analysis of cell-type abundance and reproducibility34.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [34]
The Brain Dissociation and Cell Isolation section describes the mechanical protocol and reports a higher proportion of adult mouse hippocampal neurons than with enzymatic digestion. Our copy does not establish that the cell-type-abundance comparison lacked biological replicates or could not be assessed for reproducibility. It reports four biological replicates per group for a separate proteotype analysis (Figure 3 legend), so that result does not settle the replication claim about cell-type abundance. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe inhibitor-based protocol offers greater experimental and tissue-/cell-type flexibility than previously developed experimental and computational solutions.
“Our inhibitor-based protocol offers much greater experimental and tissue-/cell-type flexibility than previously developed experimental and computational solutions for this pervasive issue (see additional discussion of some alternative methods in Supplementary Note 7)33,34,45,65,67,68.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [34]
The Discussion (page unavailable) describes limitations of earlier approaches: single-nucleus sequencing is unsuitable for some fresh-cell studies, and transcription/translation blockers do not prevent degradation of existing RNAs and proteins. Those passages provide context for comparing methods, but this paper does not evaluate the citing authors’ inhibitor-based protocol or establish that it offers greater experimental and tissue-/cell-type flexibility than previous experimental and computational solutions. (LLM, conf=0.78)
Experimental — accuracy not yet measuredTypical enzymatic digestion of mouse brain tissue induced a host of transcriptional alterations.
“Consistent with the reports from other tissues in mice, we found that typical enzymatic digestion of mouse brain tissue induced a host of transcriptional alterations34,43,44,65.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [34]
No explicit statistical methods were used to predetermine sample sizes, but the study's sample sizes exceed those reported in previous publications.
“No explicit statistical methods were used to predetermine sample sizes but our sample sizes exceed those reported in previous publications32,34,43.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [34]
The cited paper’s Introduction discusses previously published datasets, and its methods describe cell numbers and statistical analyses, but neither section compares its sample sizes with those of previous publications. The statement about not predetermining sample sizes refers to the citing authors’ own study, not to this source. Searches for sample-size comparisons and power analysis found no passage establishing the attributed comparison. (LLM, conf=0.72)
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- Authors
- Mattei, D., Ivanov, A., van Oostrum, M., Pantelyushin, S., Richetto, J., Mueller, F., Beffinger, M. M., Schellhammer, L., vom Berg, J., Wollscheid, B., Beule, D., Paolicelli, R. C., & Meyer, U.
- Affiliations
- University of Zurich (CH)
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin (DE)
- ETH Zurich (CH)
- Max Delbrück Center (DE)
- University of Lausanne (CH)
- Corresponding author
- Mattei, Daniele
- Ivanov, Andranik
- ORCID
- Venue
- International Journal of Molecular Sciences
- Publisher
- openRxiv
- Published
- 2020-05-16
- Volume
- 21
- Issue
- 21
- Pages
- 7944-7944
- ISSN
- 1422-0067 (ISSN-L)
- PMC
- PMC7663484
- OpenAlex
- W3097563614
- Cited by
- 143
- References
- 39
- Citations by year
- 2026: 18 · 2025: 19 · 2024: 25 · 2023: 28 · 2022: 32 · 2021: 21
- FWCI
- 6.65
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- International Journal of Molecular Sciences (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- SERVAL (Université de Lausanne) (publishedVersion, cc-by)
- MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
- IRIS (submittedVersion, cc-by)
- Zurich Open Repository and Archive (University of Zurich) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Repository for Publications and Research Data (ETH Zurich) (submittedVersion, cc-by)
- ETH Zürich Research Collection (cc-by)
- Zurich Open Repository and Archive (University of Zurich)
- Funders
- National Science Foundation
- Novartis Foundation
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (160259, 188524, 180099, 310030)
- Eidgenössische Technische Hochschule Zürich (grant ETH-25 15-2)
- Novartis Stiftung für Medizinisch-Biologische Forschung (#16C231)
- Universität Zürich
- Swiss Cancer Research Foundation (KFS-3852-02-2016, KFS‐4146‐02‐2017)
- European Research Council (804949)
- Functional Genomics Center Zurich
- H2020 European Research Council (804949)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- enzymatic tissue dissociation, technical bias, microglia, astrocytes, neurons, mechanical dissociation, single-cell transcriptomics, flow cytometry, mouse brain
- MeSH terms
- Animals, Astrocytes, Brain Neoplasms, Cell Separation, Chromatography, Liquid, Enzymes, Flow Cytometry, Glioma, Humans, Male, Neoplasm Transplantation, Sequence Analysis, RNA, Microglia, Gene Expression Profiling, Proteomics, Cell Line, Tumor, Mice, Tandem Mass Spectrometry, Single-Cell Analysis
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