Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Citation:

Donlin LT, Rao DA, Wei K, Slowikowski K, McGeachy MJ, Turner JD, Meednu N, Mizoguchi F, Gutierrez-Arcelus M, Lieb DJ, Keegan J, Muskat K, Hillman J, Rozo C, Ricker E, Eisenhaure TM, Li S, Browne EP, Chicoine A, Sutherby D, Noma A, Network AMPRA/SLE, Nusbaum C, Kelly S, Pernis AB, Ivashkiv LB, Goodman SM, Robinson WH, Utz PJ, Lederer JA, Gravallese EM, Boyce BF, Hacohen N, Pitzalis C, Gregersen PK, Firestein GS, Raychaudhuri S, Moreland LW, Holers VM, Bykerk V, Filer A, Boyle DL, Brenner MB, Anolik JH. Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue. Arthritis Research & Therapy 2018;20(1):139.

Abstract:

Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple highdimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of LiberaseTM TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4 and CD8 T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified.