Date: Monday, November 6, 2017
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Detailed analyses of cells from rheumatoid arthritis (RA) synovium may identify cell phenotypes and functions that drive tissue pathology and joint damage. The AMP RA/SLE network aims to deconstruct RA pathology using multiple high-dimensional analyses of cells obtained from rheumatoid synovium. These studies hold the potential to identify novel therapeutic strategies to counteract pathologic inflammatory responses in RA.
Methods: We developed a method to cryopreserve intact synovial tissue for downstream analyses of viable cells. Synovial tissue fragments were cryopreserved in a 10% DMSO-containing solution, then thawed and disaggregated. Synovial cells were analyzed using a 35-marker mass cytometry panel. In parallel, synovial cell populations, including fibroblasts, T cells, B cells, and macrophages, were flow-cytometrically sorted for low-input and plate-based single cell RNAseq transcriptomics.
Results: Intact cryopreserved synovial tissue yielded high numbers of viable cells after a thawing protocol followed by tissue dissociation. Cells isolated from cryopreserved tissue demonstrated intact expression of lineage markers by flow cytometry, comparable to freshly processed tissues. Optimization of synovial tissue dissociation performed across 6 sites, utilizing >30 arthroplasty and >20 synovial biopsy samples, yielded a consensus dissociation protocol of tissue digestion with 100ug/mL of liberase TL enzyme, which provided high cell yields, preserved surface markers, and minimized variability in RNAseq transcriptomes across replicates.
Arthroplasty samples were mechanically dissociated after enzymatic digestion by gentleMACS. To reduce cell losses when using smaller synovial biopsies, biopsy samples were mechanically dissociated by rapid, continuous magnetic stirring during enzymatic digestion and passage through an 18-gauge syringe. Cryopreserved tissue dissociated by these methods could be analyzed by multiple high-dimensional analyses. Mass cytometry revealed 1) diverse fibroblast phenotypes, 2) clear separation of memory B cells from antibody-secreting cells, and 3) multiple phenotypes of activated CD4+ and CD8+ T cells. To complement mass cytometry analysis, a flow cytometric sorting strategy was developed to collect fibroblasts, macrophages, T cells, B cells for bulk and single cell RNAseq transcriptomics. RNAseq of total synovial cells revealed a transcriptomic effect of tissue dissociation, compared to intact synovial tissue. Nonetheless, RNAseq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single cell RNAseq by CelSeq2 yielded transcriptomes of over 1000 genes/cell and demonstrated characteristic lineage markers in the expected cell populations.
Conclusion: We propose that this method of acquisition of viable cells from cryopreserved tissue can serve as a model for centralized generation of multiple, robust high-dimensional analyses of synovial samples acquired in multi-site studies. Integrated analysis of these datasets in a large patient cohort may help define the molecular heterogeneity of RA pathology and identify new therapeutic targets and biomarkers.
To cite this abstract in AMA style:Rao D, Donlin LT, Wei K, Meednu N, Turner J, McGeachy MJ, Mizoguchi F, Keegan J, Lederer J, Gutierrez-Arcelus M, Slowikowski K, Muskat K, Hillman J, Rozo C, Ricker E, Eisenhaure T, Lieb D, Li S, Browne E, Nusbaum C, Robinson WH, Kelly S, Pernis AB, Ivashkiv L, Goodman SM, Gravallese EM, Holers M, Hacohen N, Pitzalis C, Gregersen P, Bykerk VP, Moreland LW, Firestein G, Raychaudhuri S, Filer A, Boyle DL, Brenner M, Anolik JH. Methods for Generating Multiple High-Dimensional Analyses of Cryopreserved Synovial Tissue Developed By the Accelerating Medicines Partnership RA/SLE Network [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/methods-for-generating-multiple-high-dimensional-analyses-of-cryopreserved-synovial-tissue-developed-by-the-accelerating-medicines-partnership-rasle-network/. Accessed September 25, 2021.
« Back to 2017 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/methods-for-generating-multiple-high-dimensional-analyses-of-cryopreserved-synovial-tissue-developed-by-the-accelerating-medicines-partnership-rasle-network/