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Effects of Cryopreservation and Thawing on Single-Cell Transcriptomes of Human T Cells

  • Jeong Seok Lee (Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology(KAIST)) ;
  • Kijong Yi (Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology(KAIST)) ;
  • Young Seok Ju (Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology(KAIST)) ;
  • Eui-Cheol Shin (Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology(KAIST))
  • Received : 2020.06.13
  • Accepted : 2020.07.08
  • Published : 2020.08.31
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Abstract

Cryopreservation and thawing of PBMCs are inevitable processes in expanding the scale of experiments in human immunology. Here, we carried out a fundamental study to investigate the detailed effects of PBMC cryopreservation and thawing on transcriptomes. We sorted Tregs from fresh and cryopreserved/thawed PBMCs from an identical donor and performed single-cell RNA-sequencing (scRNA-seq). We found that the cryopreservation and thawing process minimally affects the key molecular features of Tregs, including FOXP3. However, the cryopreserved and thawed sample had a specific cluster with up-regulation of genes for heat shock proteins. Caution may be warranted in interpreting the character of any cluster of cells with heat shock-related properties when cryopreserved and thawed samples are used for scRNA-seq.

Keywords

Acknowledgement

This work was funded by Samsung Science and Technology Foundation under Project Number SSTF-BA1402-51 (to Shin EC).

References

  1. Cossarizza A, Chang HD, Radbruch A, Akdis M, Andra I, Annunziato F, Bacher P, Barnaba V, Battistini L, Bauer WM, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol 2017;47:1584-1797.
  2. Whitcomb BW, Perkins NJ, Albert PS, Schisterman EF. Treatment of batch in the detection, calibration, and quantification of immunoassays in large-scale epidemiologic studies. Epidemiology 2010;21 Suppl 4:S44-S50.
  3. Kim CG, Jang M, Kim Y, Leem G, Kim KH, Lee H, Kim TS, Choi SJ, Kim HD, Han JW, et al. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol 2019;4:eaay0555.
  4. Kim HD, Song GW, Park S, Jung MK, Kim MH, Kang HJ, Yoo C, Yi K, Kim KH, Eo S, et al. Association between expression level of PD1 by tumor-infiltrating CD8(+) T cells and features of hepatocellular carcinoma. Gastroenterology 2018;155:1936-1950.e17.
  5. Barcelo H, Faul J, Crimmins E, Thyagarajan B. A practical cryopreservation and staining protocol for immunophenotyping in population studies. Curr Protoc Cytom 2018;84:e35.
  6. Kreher CR, Dittrich MT, Guerkov R, Boehm BO, Tary-Lehmann M. CD4+  and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J Immunol Methods 2003;278:79-93.
  7. Wagh V, Meganathan K, Jagtap S, Gaspar JA, Winkler J, Spitkovsky D, Hescheler J, Sachinidis A. Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing. Stem Cell Rev Rep 2011;7:506-517.
  8. Papalexi E, Satija R. Single-cell RNA sequencing to explore immune cell heterogeneity. Nat Rev Immunol 2018;18:35-45.
  9. Madissoon E, Wilbrey-Clark A, Miragaia RJ, Saeb-Parsy K, Mahbubani KT, Georgakopoulos N, Harding P, Polanski K, Huang N, Nowicki-Osuch K, et al. scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation. Genome Biol 2019;21:1.
  10. Patel AP, Tirosh I, Trombetta JJ, Shalek AK, Gillespie SM, Wakimoto H, Cahill DP, Nahed BV, Curry WT, Martuza RL, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 2014;344:1396-1401.
  11. Szabo PA, Levitin HM, Miron M, Snyder ME, Senda T, Yuan J, Cheng YL, Bush EC, Dogra P, Thapa P, et al. Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease. Nat Commun 2019;10:4706.
  12. Tikhonova AN, Dolgalev I, Hu H, Sivaraj KK, Hoxha E, Cuesta-Dominguez A, Pinho S, Akhmetzyanova I, Gao J, Witkowski M, et al. The bone marrow microenvironment at single-cell resolution. Nature 2019;569:222-228.
  13. Tran HT, Ang KS, Chevrier M, Zhang X, Lee NY, Goh M, Chen J. A benchmark of batch-effect correction methods for single-cell RNA sequencing data. Genome Biol 2020;21:12.
  14. Guillaumet-Adkins A, Rodriguez-Esteban G, Mereu E, Mendez-Lago M, Jaitin DA, Villanueva A, Vidal A, Martinez-Marti A, Felip E, Vivancos A, et al. Single-cell transcriptome conservation in cryopreserved cells and tissues. Genome Biol 2017;18:45.
  15. Wohnhaas CT, Leparc GG, Fernandez-Albert F, Kind D, Gantner F, Viollet C, Hildebrandt T, Baum P. DMSO cryopreservation is the method of choice to preserve cells for droplet-based single-cell RNA sequencing. Sci Rep 2019;9:10699.
  16. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 2018;36:411-420.