Abstract
BackgroundThe purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to RPMI to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders.MethodsWe performed single-cell RNA sequencing on paired skin biopsy specimens from 3 patients with LS. Each patient with one sample cryopreserved in CryoStor® CS10 and one fresh in RPMI media using 10× Genomics sequencing.ResultsLevels of cell viability and yield were comparable between CryoStor® CS10 (frozen) and RPMI (fresh) preserved cells. Furthermore, gene expression between preservation methods was collectively significantly correlated and conserved across all 18 identified cell cluster populations.ConclusionComparable cell population and transcript expression yields between CryoStor® CS10 and RPMI preserved cells support the utilization of cryopreserved skin tissue in single-cell analysis. This suggests that employing standardized cryopreservation protocols for the skin tissue will help facilitate multi-site collaborations looking to identify mechanisms of disease in disorders characterized by cutaneous pathology.
Highlights
The purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to Roswell Park Memorial Institute (RPMI) to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders
Our objective was to determine whether the use of CryoStor® CS10 produced adequate preservation of tissue samples and the cell types of interest allowing for the use of frozen samples for large-scale studies in localized scleroderma skin, as well as other skin disorders that benefit from multi-institutional collaborations
The average depth of the biopsies was 3.4 mm. These two biopsies were allocated for scRNAseq; one whole biopsy was placed in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco®, Gaithersburg, MD) and put on ice while the other whole biopsy was placed in chilled CryoStor® CS10 cell preservation media (BioLife Solutions®, Bothell, WA), incubated at 4 °C for 10 min put on dry ice per manufacturer’s instructions
Summary
The purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to RPMI to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders. Our laboratory has an interest in the examination of the transcriptome in the localized scleroderma (morphea) skin using a high-resolution scRNAseq technology with advanced bioinformatics to better characterize the dysregulation of the IFN-γ pathway and contributing cell types that are likely relevant to disease propagation [7,8,9,10,11,12]. To study the skin transcriptomic immunophenotype across the clinical spectrum of localized scleroderma (LS), we formulated a dermatology-rheumatology partnership across two academic centers with robust LSspecific cohorts. These registries, the National Registry of Childhood Onset Scleroderma (NRCOS) and the Morphea in Adults and Children (MAC), provide a rich resource of well-phenotyped patients interested in providing skin biopsy samples for our research protocols. The overall intention is that these validation steps for proper sample procurement of skin for scRNAseq will set the stage for our larger study in localized scleroderma (morphea), and serve as a platform for technical reference and cell type preservation for other rare autoimmune skin diseases, such as cutaneous lupus erythematosus and dermatomyositis, in which obtaining materials across multiple centers as a consortium would be beneficial
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