Effects of enzymatic digestion, cell culture and preservation conditions on surface CD62L expression of primary murine CD3+CD4+ T cells

Liu, Qi; Zhao, Ruihua; Huang, Chun Ming; Zhou, Kai; Zhang, Xiao Lian; Pan, Quanjun

doi:10.4066/biomedicalresearch.29-17-3070

Cited by 9 publications

(8 citation statements)

References 24 publications

(35 reference statements)

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“…The incorporation of heat or enzymes to enable this dissociation, however, is almost universal (27). The treatment of tissues with enzymes, heat, or a combination of the two can impact the resulting cell dissociations in ways that may influence conclusions drawn from surrounding research (12).…”

Section: Discussionmentioning

confidence: 99%

“…For both methods, single cell suspensions are required as input material for proper staining and analysis and to prevent instrument clogging. While enzyme and heat-based dissociation protocols have often preceded flow cytometry and FACS, recent studies have demonstrated a significant loss of certain immunological cell markers following dissociation with enzymes (12,13). Further, single-cell analysis methods often target rare cell types, making the accurate preservation of varied cell populations and cell markers essential for these applications.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An enzyme-free, cold-process acoustic method for gentle and effective tissue dissociation

MacMullan,

Busalacchi,

Quisling

et al. 2023

Preprint

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As biological advances continue to improve the resolution of genomic and proteomic studies, the quality of single cell suspensions is becoming increasingly important. While conventional approaches use enzymes which may require heat Abbreviations: Bulk Lateral Ultrasound (BLU) activation to break down extracellular tissue matrices and gain access to single cells, recent studies have suggested that these harsh biochemical and heat-based treatments may result in genomic and proteomic modulation. To minimize these dissociation artifacts, we have developed an instrument for dissociating cells from various tissue matrices using Bulk Lateral Ultrasound (BLU™) energy. This enzyme-free, gentle mechanical dissociation maintains sample temperatures below 8°C for the duration of processing, resulting in high-fidelity single cell suspensions with comparable viability and live cell counts to those obtained with conventional enzymatic dissociations. Here, in murine-derived brain, heart, lung, and B16 melanoma tumor tissue dissociated by either BLU or by a commercially available dissociation kit which uses enzymes and heat, we compare cell viability and expression of population-specific immunological markers. The dramatic differences observed in cell surface expression suggest that cells dissociated using enzymes and heat may be experiencing stress-induced changes post-harvest that could impact conclusions and impede research progress. Alternatively, using gentle mechanical dissociation with BLU, we demonstrate the preservation of these markers and enable a minimally invasive alternative to obtaining high integrity single cell suspensions.HighlightsNovel, acoustic energy-based, enzyme-free dissociation improves single cell suspensionsEnzymatic dissociation diminishes cell counts, viability, and surface marker expressionImmunophenotyping reveals marker preservation by acoustic-based dissociaton

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An enzyme-free, cold-process acoustic method for gentle and effective tissue dissociation

MacMullan,

Busalacchi,

Quisling

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…However, if the combination of mechanical dissociation and collagenase treatment is used, the concentration or incubation time of collagenase should be reduced, since the initial mechanical dissociation would increase the surface area of the tissue, allowing quicker digestion. Unregulated exposure of tissues to collagenase can affect the expression of certain molecules on the cell surface of leukocytes [28,29] ( Figure 2). Similarly, it has also been suggested that enzymatic digestion could alter or damage the T cell receptor (TCR) on T cells [30].…”

Section: Sample Processing and Preparation Of Single-cell Suspensionmentioning

confidence: 99%

Technical Considerations in Ex Vivo Human Regulatory T Cell Migration and Suppression Assays

Irekeola

Lazim

et al. 2020

Cells

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Regulatory T cells (Tregs) are renowned for maintaining homeostasis and self-tolerance through their ability to suppress immune responses. For over two decades, Tregs have been the subject of intensive research. The immunosuppressive and migratory potentials of Tregs have been exploited, especially in the areas of cancer, autoimmunity and vaccine development, and many assay protocols have since been developed. However, variations in assay conditions in different studies, as well as covert experimental factors, pose a great challenge to the reproducibility of results. Here, we focus on human Tregs derived from clinical samples and highlighted caveats that should be heeded when conducting Tregs suppression and migration assays. We particularly delineated how factors such as sample processing, choice of reagents and equipment, optimization and other experimental conditions could introduce bias into the assay, and we subsequently proffer recommendations to enhance reliability and reproducibility of results. It is hoped that prioritizing these factors will reduce the tendencies of generating false and misleading results, and thus, help improve our understanding and interpretation of Tregs functional studies.

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“…In addition, animal-based enzymes were found to decrease the expression levels of proteins related to growth and metabolism, and to increase the expression levels of proteins related to apoptosis. Liu et al found that the use of trypsin detachment could also affect the autoimmune function of primary cells [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes

Huang

et al. 2022

Cells

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Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes.

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Effects of enzymatic digestion, cell culture and preservation conditions on surface CD62L expression of primary murine CD3+CD4+ T cells

Cited by 9 publications

References 24 publications

An enzyme-free, cold-process acoustic method for gentle and effective tissue dissociation

An enzyme-free, cold-process acoustic method for gentle and effective tissue dissociation

Technical Considerations in Ex Vivo Human Regulatory T Cell Migration and Suppression Assays

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes

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