Isolation and culture of murine aortic cells and RNA isolation of aortic intima and media: Rapid and optimized approaches for atherosclerosis research. (April 2022)

• Record Type: Journal Article
• Authors: Chen, Jingshu; Zhuang, Rulin; Cheng, Henry S.; Jamaiyar, Anurag; Assa, Carmel; McCoy, Michael; Rawal, Shruti; Pérez-Cremades, Daniel; Feinberg, Mark W.
• Is Part Of: Atherosclerosis. Volume 347(2022)
• Page Start: 39
• Page End: 46
• Publication Date: 2022-04
• Subjects: Atherosclerosis -- Aortic primary cells -- Magnetic bead cell isolation -- Intima peeling; Arteriosclerosis -- Periodicals; Electronic journals; 616.136
• DOI: 10.1016/j.atherosclerosis.2022.03.011
• Languages: English
• ISSNs: 0021-9150

Abstract

Abstract: Background and aims: Isolation of cellular constituents from the mouse aorta is commonly used for expression or functional analyses in atherosclerosis research. However, current procedures to isolate primary cells are difficult, inefficient, and require separate mice. RNA extraction from aortic intima and media for transcriptomic analysis is also considered difficult with mixed RNA yields. To address these gaps, we provide: 1) a rapid, efficient protocol to isolate and culture diverse cell types concomitantly from the mouse aorta using immunomagnetic cell isolation; and 2) an optimized aortic intimal peeling technique for efficient RNA isolation from the intima and media. Methods and results: Aortic cells were obtained using an enzymatic solution and different cell types were isolated by magnetic beads conjugated to antibodies targeting endothelial cells (CD31 + ), leukocytes (CD45 + ), and fibroblast cells (CD90.2+), and smooth muscle cells were isolated by negative selection. Our protocol allows the isolation of relatively large numbers of cells (10, 000 cells per aorta) in a predictable manner with high purity (>90%) verified by cell-marker gene expression, immunofluorescence, and flow cytometry. These cells are all functionally active when grown in cell culture. We also provide a rapid method to collect aortic intima-enriched RNA from Ldlr −/− mice utilizing an intima peeling approach and assess transcriptomic profiling associated with accelerated lesion formation. Conclusions: This protocol provides an effective means for magnetic bead-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic studies for a range of vascular diseases including atherosclerosis. Graphical abstract: Image 1 Highlights: We provide an optimized enzymatic digestion protocol of the aorta, ideal for scRNAseq, FACS, and cell isolation and culture. The new magnetic beads-based selection method is rapid and efficiently isolates diverse aortic cells simultaneously. Isolated primary aortic ECs, SMCs, leukocytes, and fibroblasts can be cultured for further vascular biology experiments. An optimized intima peeling approach enables efficient RNA enrichment specifically from the aortic intima or media.