Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

Abstract

BackgroundDetailed 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 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.MethodsMultiple 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.ResultsUpon 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 Liberase™ 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1631-y) contains supplementary material, which is available to authorized users.

HBEGF+macrophages identified in rheumatoid arthritis promote joint tissue invasiveness and are reshaped differentially by medications

“…To examine the impact of therapeutically targeting HBEGF + macrophages in RA joints, we directly assayed synovial tissue from RA patients with definitive RA diagnoses ( 40, 41 ) and extensive joint inflammation confirmed by histologic scoring ( 42, 43 ) (Table S1, n=8). Specifically, in an ex vivo tissue assay, synovium was dissociated into cell suspensions ( 44 ) and cultured with FDA-approved RA medications (Fig. 4B).…”

“…The multicenter RA/SLE Network of the Accelerating Medicines Partnership (AMP) consortium enrolled individuals meeting the ACR 2010 RA classification according to protocols approved by the institutional review board at each site ( 22, 44 ). Synovial tissues were collected from ultrasound-guided biopsies or joint replacement surgery and viably frozen in Cryostor CS10 cryopreservation media (Sigma-Aldrich).…”

Versatile workflow for cell type resolved transcriptional and epigenetic profiles from cryopreserved human lung

“…Using established cell lines and isolated primary cells, several studies demonstrated that cell cryopreservation does not alter global transcriptional profiles of cells 23,42,43 . In addition, two studies combined cryopreservation of human tissue (ovarian cancers (Guillaumet-Adkins, 2017 #72)) and human synovial tissue 44 ) with subsequent single-cell RNA-seq analysis. We extend these observations to lung tissue and show that cryopreservation of human lung parenchyma and lung tumor pieces is also compatible with downstream processing using the 10x Genomics single-cell profiling platform.…”

Tissue‐Resident Memory T Cells: Sequestered Immune Sensors and Effectors of Inflammation in Spondyloarthritis

“…In rheumatoid arthritis (RA), analysis of synovial tissue with single‐cell RNA sequencing and cytometry by time‐of‐flight mass spectrometry have revealed discrete cell subpopulations with transcriptomic and cell surface expression profiles linked to functional activities . These analyses, focused on the synovium and not the peripheral blood, provided new insights regarding key pathogenic cell–cell interactions and underscore the necessity of examining cellular and molecular events in the target tissue.…”