Ahmad Herra, Gopakumar Jayakrishnan, Nachun Daniel C, Ma Lisa, D'Addabbo Jessica, Huang Xianxi, Koyano Tiffany, Boyd Jack, Woo Yi-Ping Joseph, Fong Robyn, Aalami Oliver, Nguyen Patricia K, Jaiswal Siddhartha
Department of Pathology, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA.
Department of Cardiology, Charité Universitätsmedizin, Hindenburgdamm 30, 12203 Berlin, Germany.
Cardiovasc Res. 2025 Apr 29;121(3):396-404. doi: 10.1093/cvr/cvaf014.
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.
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.
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.
单细胞RNA测序（scRNA-seq）是探索人类动脉粥样硬化斑块内细胞异质性的有力方法，但通常需要新鲜组织来保持细胞膜完整性，这限制了大规模生物样本库用于后续分析的可行性。本研究的目的是确定脆弱和坏死的动脉粥样硬化斑块组织的冷冻保存是否会影响后续scRNA-seq分析中造血细胞的活力和转录组图谱，从而使冷冻保存的动脉粥样硬化斑块样本可用于未来研究。
我们对五对新鲜和冷冻保存的动脉粥样硬化斑块样本进行了scRNA-seq分析，其中三对来自冠状动脉，两对来自颈动脉。每个样本经酶消化后，分选CD45+造血细胞，并使用10×基因组学scRNA-seq工作流程进行处理。每个样本的一半立即进行处理，另一半在液氮中冷冻保存平均5周后解冻并处理。在颈动脉样本中，我们注意到缺乏LYVE1+巨噬细胞，这可能是由于内膜切除术过程中外膜层的丢失。我们的结果表明，冷冻保存对细胞完整性有适度影响，导致冷冻样本中线粒体RNA的相对丰度增加。在造血群体中，新鲜样本和冷冻保存样本在独特检测的转录本、细胞聚类或转录图谱方面观察到的差异极小。
我们的研究表明，冷冻保存的人类动脉粥样硬化斑块样本可以通过scRNA-seq成功进行分析，转录组数据与新鲜样本相当。这些发现表明，冷冻保存是一种可行的动脉粥样硬化斑块组织生物样本库方法，有助于大规模研究，而无需立即进行样本处理。
