Optimisation of a novel single cell transcriptomic platform utilising cryopreservation of bronchial mucosal cells in asthma

Résumé fourni par la source

Single cell RNA sequencing (scRNAseq) of mucosal biopsies offers opportunities to understand immune heterogeneity in inflammatory disease; however, methodology for digestion and preservation is not standardised and few studies have reported biopsy cell counts. We sought to assess the quality of HIVE CLX™, a novel scRNAseq platform utilising longitudinal cryopreserved digested bronchial mucosal cells and identify changes in transcriptome phenotypes after ICS exposure. Three bronchial biopsies were each obtained from 5 patients of a phase IV interventional asthma study (MAELABA; NCT04503460 ). Samples from 3 of the 5 patients were paired, taken at bronchoscopies before and after 2 weeks of combination ICS/LABA. Fresh tissue digestion was performed on 3 pooled bronchial samples using an adapted protocol pre-validated in-house. Following cell capture and cryopreservation, transcriptome recovery and library preparation were performed prior to bioinformatic analysis. Mean cell counts and median cryopreservation storage time of the pooled intrasubject samples were 151.9±100.5 cells/µL and 59.5±39.4 days respectively. Azimuth and SingleR were used to identify 12 distinct cell populations including 782 ciliated, 159 CD4+ & 131 CD8+ T cells, 50 B cells, 74 macrophage/DCs and 9 mast cells (all cells/µL). Due to high level of RNases, no significant eosinophil data could be derived, and more tissue is required for rare cell (e.g. ILC2) phenotyping. We have demonstrated digested bronchial mucosal tissue retains transcriptomic phenotyping potential after cryopreservation using HIVE CLX™, highlighting its utility as a viable platform in multicentre translational research studies.

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