Single cell RNA sequencing of haematopoietic cells in fresh and frozen human atheroma tissue

Abstract Aims Single-cell RNA sequencing (scRNA-seq) is a powerful method for exploring the cellular heterogeneity within human atheroma but typically requires fresh tissue to preserve cell membrane integrity, limiting the feasibility of large-scale biobanking for later analysis. The aim of this study was to determine whether cryopreservation of fragile and necrotic atheroma tissue affects the viability and transcriptomic profiles of haematopoietic cells in subsequent scRNA-seq analysis, enabling the use of cryopreserved atheroma samples for future research. Methods and results We performed scRNA-seq on five paired fresh and cryopreserved atheroma samples—three from coronary arteries and two from carotid arteries. Each sample was enzymatically digested, sorted for CD45+ haematopoietic cells, and processed using the 10× Genomics scRNA-seq workflow. Half of each sample was processed immediately, while the other half was cryopreserved in liquid nitrogen for an average of 5 weeks before thawing and processing. In carotid artery samples, we noted the absence of LYVE1+ macrophages, likely due to the loss of the adventitial layer during endarterectomy procedures. Our results indicated that cryopreservation modestly affected cellular integrity, leading to an increase in the relative abundance of mitochondrial RNA in frozen samples. Minimal differences were observed between fresh and cryopreserved samples in uniquely detected transcripts, cell clustering, or transcriptional profiles within haematopoietic populations. Conclusions Our study demonstrates that cryopreserved human atheroma samples can be successfully profiled using scRNA-seq, with comparable transcriptomic data to that obtained from fresh samples. These findings suggest that cryopreservation is a viable method for biobanking atheroma tissues, facilitating large-scale studies without the need for immediate sample processing.