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High-dimensional immune phenotyping and transcriptional analyses reveal robust recovery of viable human immune and epithelial cells from frozen gastrointestinal tissue

Abstract

Simultaneous analyses of peripheral and mucosal immune compartments can yield insight into the pathogenesis of mucosal-associated diseases. Although methods to preserve peripheral immune cells are well established, studies involving mucosal immune cells have been hampered by lack of simple storage techniques. We provide a cryopreservation protocol allowing for storage of gastrointestinal (GI) tissue with preservation of viability and functionality of both immune and epithelial cells. These methods will facilitate translational studies allowing for batch analysis of mucosal tissue to investigate disease pathogenesis, biomarker discovery and treatment responsiveness.

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References

  1. 1.

    Neurath, M. F. Current and emerging therapeutic targets for IBD. Nat. Rev. Gastroenterol. Hepatol. 14, 269–78 (2017).

  2. 2.

    Starr, A. E. et al. Proteomic analysis of ascending colon biopsies from a paediatric inflammatory bowel disease inception cohort identifies protein biomarkers that differentiate Crohn’s disease from UC. Gut 66, 1573–83 (2017).

  3. 3.

    Barnes, E. L., Liew, C. C., Chao, S. & Burakoff, R. Use of blood based biomarkers in the evaluation of Crohn’s disease and ulcerative colitis. World J. Gastrointest. Endosc. 7, 1233–37 (2015).

  4. 4.

    Burakoff, R. et al. Blood-based biomarkers used to predict disease activity in Crohn’s disease and ulcerative colitis. Inflamm. Bowel Dis. 21, 1132–40 (2015).

  5. 5.

    Bendall, S. C., Nolan, G. P., Roederer, M. & Chattopadhyay, P. K. A deep profiler’s guide to cytometry. Trends Immunol. 33, 323–32 (2012).

  6. 6.

    Bendall, S. C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–96 (2011).

  7. 7.

    Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J. & Nolan, G. P. Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry A 81, 552–66 (2012).

  8. 8.

    Levine, J. H. et al. Data-driven phenotypic dissection of aml reveals progenitor-like cells that correlate with prognosis. Cell 162, 184–97 (2015).

  9. 9.

    Amir el, A. D. et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat. Biotechnol. 31, 545–52 (2013).

  10. 10.

    Mirza, A. H. et al. Transcriptomic landscape of lncRNAs in inflammatory bowel disease. Genome Med. 7, 39 (2015).

  11. 11.

    Reardon, A. J., Elliott, J. A. & McGann, L. E. Investigating membrane and mitochondrial cryobiological responses of HUVEC using interrupted cooling protocols. Cryobiology 71, 306–17 (2015).

  12. 12.

    Keane, K. N., Calton, E. K., Cruzat, V. F., Soares, M. J. & Newsholme, P. The impact of cryopreservation on human peripheral blood leucocyte bioenergetics. Clin. Sci. 128, 723–33 (2015).

  13. 13.

    Hughes, S. M. et al. Cryopreservation of human mucosal leukocytes. PLoS ONE 11, e0156293 (2016).

  14. 14.

    Nishimura, M., Mitsunaga, S. & Juji, T. Frozen-stored granulocytes can be used for an immunofluorescence test to detect granulocyte antibodies. Transfusion 41, 1268–72 (2001).

  15. 15.

    Lionetti, F. J., Hunt, S. M., Lin, P. S., Kurtz, S. R. & Valeri, C. R. Preservation of human granulocytes. II. Characteristics of granulocytes obtained by counterflow centrifugation. Transfusion 17, 465–72 (1977).

  16. 16.

    Frim, J. & Mazur, P. Approaches to the preservation of human granulocytes by freezing. Cryobiology 17, 282–6 (1980).

  17. 17.

    Rowe, A. W. & Lenny, L. L. Cryopreservation of granulocytes for transfusion: studies on human granulocyte isolation, the effect of glycerol on lysosomes, kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol. Cryobiology 17, 198–12 (1980).

  18. 18.

    Baust, J. M., Corwin, W., Snyder, K. K., Van Buskirk, R. & Baust, J. G. Cryopreservation: evolution of molecular based strategies. Adv. Exp. Med. Biol. 951, 13–29 (2016).

  19. 19.

    Sato, T. & Clevers, H. Primary mouse small intestinal epithelial cell cultures. Methods Mol. Biol. 945, 319–28 (2013).

  20. 20.

    Zhao, S. et al. QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization. BMC Genomics 17, 39 (2016).

  21. 21.

    Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).

  22. 22.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

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Acknowledgements

L.K. is a recipient of a Career Development Award Grant from the Crohn’s and Colitis Foundation of America (CDA 422348). S.B.S. is supported by NIH Grants: R01 DK115217, R56 AI125766; P30DK034854, the Helmsley Charitable Trust, and the Wolpow Family Chair in IBD Treatment and Research. C.R. is supported by NIH K08 DK106562-01A1. D.T.B. is supported by R01DK084056, the Timothy Murphy Fund, the IDDRC P30HD18655 and the HDDC P30DK034854. V.M. is supported by 5 T32 DK7533

Authors contributions

L.K., G.B., A.R., J.C., S.W., V.M., and M.F. performed the CyTOF experiments. L.K., G.B., and A.R. analyzed the CyTOF data. C.R. and F.Z. performed the enteroid experiments. W.G. and S.J. performed and analyzed the RNAseq data. J.L. provided the RNAseq samples and analyzed the data. V.T.T. performed and analyzed the FACS experiments. J.D.G. provided BCH samples. M.B. provided the Penn samples. L.K., G.B., A.R., J.C., W.G., V.M., D.T.B., M.M., and S.B.S. conceived the experiments and analyzed the data. L.K. and S.B.S. wrote the manuscript.

Author information

Competing interests

Conflicts of Interest: S.B.S. is supported by grants or in-kind contributions from Pfizer, Janssen, Merck, and Regeneron. He is on the scientific advisory boards of Pfizer, Janssen, IFM Therapeutics, Lycera, Inc., Celgene, Pandion Therapeutics, and Applied Molecular Transport. He has consulted for Amgen and Hoffman La-Roche. Except for support from Pfizer that performed the RNA seq experiments in this work and assisted in the analytics, there are no conflicts of interest that are related to this work.

Correspondence to Scott B. Snapper.

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