A standardized approach for the isolation of vascular smooth muscle cells from carotid atherosclerotic plaques.

Vascular smooth muscle cells (VSMCs) are critical players in atherosclerotic plaque development and progression, but existing isolation methods often yield low cell viability, making them suboptimal for several downstream applications. As such, we aimed to optimize an enzymatic digestion protocol for isolating viable VSMCs from human carotid plaques suitable for cell culture analysis. Carotid plaques were obtained following endarterectomy and subjected to a streamlined enzymatic digestion protocol using a simplified cocktail optimized to minimize cell damage. After brief culture to passage 1, single-cell suspensions were processed using the 10X Genomics Chromium platform. Single-cell RNA sequencing (scRNA-seq) data underwent rigorous quality filtering, Harmony-based integration, clustering, gene ontology (GO) enrichment analysis, and pseudotime trajectory inference to validate cell identity and assess phenotypic transitions. The protocol yielded high-viability single-cell suspensions, with approximately 23,661 cells sequenced after quality filtering from four carotid plaques: two classified as stable and two as unstable, obtained from both male and female patients. scRNA-seq revealed that the majority of isolated cells were VSMCs, confirmed through positive module scores for canonical VSMC markers (TAGLN, ACTA2, MYH11, CNN1, MYL9) and GO enrichment of VSMC-related biological processes. Label transfer analysis using the Tabula Sapiens vasculature reference further confirmed VSMCs as the predominant population. Subcluster analysis identified four transcriptionally distinct VSMC phenotypes. Pseudotime analysis demonstrated a trajectory originating from contractile VSMCs, progressing through intermediate foam cell-like and inflammatory synthetic states, and ultimately transitioning into fibroblast-like phenotypes. This confirms that the entire isolated population was VSMC-derived and undergoing phenotypic switching. In conclusion, this optimized protocol enables the reliable isolation of highly viable VSMCs from human carotid plaques, as validated by single-cell transcriptomic profiling. These findings lay the groundwork for future applications of isolated VSMCs in single-cell studies, disease modeling, and mechanistic investigations of VSMC phenotypic switching and plaque instability.