High dimensional analyses of cells dissociated from cryopreserved synovial tissue
Authors: Donlin Laura T. , Rao Deepak A. , Wei Kevin , Slowikowski Kamil , McGeachy Mandy J. , Turner Jason D. , Meednu Nida , Mizoguchi Fumitaka , Gutierrez-Arcelus Maria , Lieb David J. , Keegan Joshua , Muskat Kaylin , Hillman Joshua , Rozo Cristina , Ricker Edd , Eisenhaure Thomas M. , Li Shuqiang , Browne Edward P. , Chicoine Adam , Sutherby Danielle , Noma Akiko ,   Accelerating Medicines Partnership: RA/SLE Network, Nusbaum Chad , Kelly Stephen , Pernis Alessandra B. , Ivashkiv Lionel B. , Goodman Susan M. , Robinson William H. , Utz Paul J. , Lederer James A. , Gravallese Ellen M. , Boyce Brendan F. , Hacohen Nir , Pitzalis Costantino , Gregersen Peter K. , Firestein Gary S. , Raychaudhuri Soumya , Moreland Larry W. , Michael Holers V. , Bykerk Vivian P. , Filer Andrew , Boyle David L. , Brenner Michael B. , Anolik Jennifer H. 
Published: March 2018

Background

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 (AMP) RA/SLE network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional 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 100µg/mL of Liberase TL™ enzyme. 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: 1) diverse fibroblast phenotypes, 2) distinct populations of memory B cells and antibody-secreting cells, and 3) multiple CD4+ and CD8+ T cell activation states. Bulk RNA sequencing 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.

Conclusion

We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.