Eliminating artifacts in tissue dissociation methods: A Review

Tissue dissociations methods determine both the cell quantity and quality of resulting single-cell suspensions. Prevalent protocols which use enzymes activated by heat can cause dissociation artifacts, artificially skewing the results of flow cytometry and single-cell RNA sequencing (1,2). The Cellsonics SimpleFlowTM which uses Bulk Lateral Ultrasonic (BLUTM) energy offers an enzyme and heat-free alternative for tissue dissociation. When tested in parallel with enzyme and heat dissociation protocols, BLU is found to be superior for examining cell surface markers on lung, heart, brain, and tumor tissues.

In these studies, conducted by researchers at Grand Valley State University, BLU dissociation resulted in a greater number of cells of interest across tissue types. They demonstrated more live CD8+ T cells were isolated from murine tumor, lung, cardiac, and brain samples using BLU than a commercially available enzyme and heat-based dissociation kit (3,4). In heart tissue, significantly increased lymphocyte, macrophage, antigen-presenting cell, dendritic cell, endothelial cell, and myelinogenic progenitor cell surface markers (CDs) were observed on BLU dissociated cells than on those treated with enzymes and heat (5). Compared to the enzyme and heat-based protocol, brain cells dissociated by BLU had higher expression of CD86 and Ly6G, markers that may be used to distinguish resident microglia and infiltrating neutrophils (6,7). These findings suggest that gentle mechanical dissociation by BLU preserves more CDs, enabling detailed analysis of inflammation and disease states. The results also suggest that enzyme and heat dissociation techniques may be misrepresentative of in vivo tissue environments.

Concern about the effects of enzymes and heat on living cells is supported in scientific literature, as heat, the use of enzymes, and the longer dissociation times required by these methods have all been shown to alter CD and gene expression by inducing a stress response (8,9). In a 2016 study by Lacar et al. where single nucleus RNA sequencing was employed to compare the transcriptomes of dissociated and whole tissue neurons, researchers found that, following enzymatic digestion, inactivated neurons appeared activated. They demonstrated that protease dissociation mimicked the immediate early genes (IEGs) Fos, Arc, and Egr1 seen in activated neuron transcriptomes (10). A 2021 review published by Machado et al. highlighted that dissociation-induced IEG expression was also observed in murine visual cortex, kidney, liver, and skeletal muscle, demonstrating enzymatic dissociation artifacts occur across multiple tissue types (11). Another work from Machado et al. found a “strong correlation” between long dissociation protocol times and the stress index experienced by the cell (8). A comparison of dissociation techniques by Mattei et al. found more accurate cell population ratios isolated by cold mechanical dissociation than by heated and enzyme treated brain cells (2). Additionally, enzyme dissociation methods were found to induce deregulation in genes of brain cells via RNA-editing, protein translation, and changes to metabolic functions.

Both flow cytometry and single cell sequencing analysis methods are optimized by inputting cells with heterogeneity matching that of living and diseased tissue. Therefore, the exponential growth of the single cell workflows necessitates high quality single cell suspensions with minimal dissociation artifacts (12). To enable high quality single cell analysis, an upstream tissue dissociation method that maintains immune cell markers and an accurate single cell transcriptome is essential for ensuring that discoveries made are free of misrepresentative dissociation artifacts (13). Due to the changes in recovered cell types and gene and CD expression, dissociation with a heat and enzyme free method such as BLU energy, which preserves these attributes, is more suitable than the use of heat and enzyme dissociation for these applications.

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  1. Panchision DM, Chen HL, Pistollato F, Papini D, Ni HT, Hawley TS. Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Novel Functional Relationships Among Cells Expressing CD133, CD15, and CD24. STEM CELLS. 2007;25(6):1560–70.
  2. Mattei D, Ivanov A, van Oostrum M, Pantelyushin S, Richetto J, Mueller F, et al. Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations. Int J Mol Sci. 2020 Jan;21(21):7944.
  3. Significantly Improved CD8+ T Cell Isolation From Mouse Tumors With SimpleFlow [Internet]. 2023 [cited 2023 Sep 15]. Available from: https://blog.cellsonics.com/blog/streamlining-mouse-tumor-dissociation
  4. Enzyme-free dissociation preserves critical mouse lung cell markers [Internet]. 2023 [cited 2023 Sep 19]. Available from: https://blog.cellsonics.com/blog/mouse-lung
  5. Retain Critical Mouse Cardiac Markers With Cellsonics SimpleFlow [Internet]. 2023 [cited 2023 Sep 19]. Available from: https://blog.cellsonics.com/blog/mouse-cardiac-markers
  6. Enhanced Mouse Brain Cell Surface Expression With SimpleFlow BLU [Internet]. 2023 [cited 2023 Sep 15]. Available from: https://blog.cellsonics.com/blog/mousebrain
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  9. Denisenko E, Guo BB, Jones M, Hou R, de Kock L, Lassmann T, et al. Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biol. 2020 Jun 2;21(1):130.
  10. Lacar B, Linker SB, Jaeger BN, Krishnaswami S, Barron J, Kelder M, et al. Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat Commun. 2016 Apr 19;7:11022.
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  12. Zappia L, Theis FJ. Over 1000 tools reveal trends in the single-cell RNA-seq analysis landscape. Genome Biol. 2021 Oct 29;22(1):301.
  13. Reichard A, Asosingh K. Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry. Cytom Part J Int Soc Anal Cytol. 2019 Feb;95(2):219–26.