A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature

• Published in: Cell reports methods Vol. 3; no. 3; p. 100431
• Main Authors: Bjørnholm, Katrine Dahl, Del Gaudio, Francesca, Li, Hao, Li, Weihan, Vazquez-Liebanas, Elisa, Mäe, Maarja Andaloussi, Lendahl, Urban, Betsholtz, Christer, Nilsson, Per, Karlström, Helena, Vanlandewijck, Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.03.2023; Elsevier
• Subjects: blood-brain barrier; capillary; endothelial cell; microvascular isolation; neurovascular unit; pericyte; single-cell RNA sequencing; vasculature; single-cell RNA sequencing; blood-brain barrier; vasculature; pericyte; capillary; CP: Neuroscience; endothelial cell; microvascular isolation; neurovascular unit
• DOI: 10.1016/j.crmeth.2023.100431
• ISSN: 2667-2375

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture. [Display omitted] •We develop a method to isolate brain vasculature for ex vivo and single-cell studies•The cellular diversity of the neurovascular unit is preserved•The method avoids the need for transgenic reporters•An online browsing tool is available for gene-by-gene investigation of cell types Single-cell analysis of vascular cells in the brain is challenging due to the low abundance of these cells relative to neuronal and glial cells.1 Previous investigations have successfully used reporter mouse strains, in which labeling of specific cell types in the neurovascular unit was exploited to isolate the cells using fluorescence-activated cell sorting (FACS).2 The generation and use of mouse reporter strains is, however, a slow and expensive process, especially if investigations are to be carried out in several different genetic mouse strains or transgenic disease models that, in turn, need to be combined with transgenic reporters for multiple vascular cell types. Moreover, FACS subjects cells to fluctuations in pressure, shape, and temperature, which may cause stress or injury-induced responses.3 Endothelial cells, as well as vascular smooth muscle cells, are very sensitive to changes in pressure since they need to respond rapidly to changes in blood flow.4 While it has been shown that the sorting process may only induce small changes in expression pattern in epithelial cells after FACS, genes related to the stimulation of angiogenic pathways were highly susceptible to changes in expression after FACS,5 underlining the importance of avoiding this method when investigating vascular cells. This is less problematic when single cells are directly sorted into lysis buffer compared with when cell suspensions are created for droplet encapsulation, which takes a longer time, thereby allowing a FACS-induced injury to influence the cell’s mRNA content. To overcome these current experimental hurdles, we have developed a magnetic bead-based protocol that enables isolation of microvascular segments and applied this to single-cell RNA sequencing, as well as protein chemistry and cell culture. The protocol enables analysis of the vasculature at the single-cell level in any mouse model, alleviating the need for mouse reporter cell lines and FACS prior to analysis. Bjørnholm et al. have developed a simple and robust brain vascular isolation protocol suitable for different types of experiments. Their protocol allows researchers to focus selectively on the cellular components of the blood-brain barrier, such as endothelial cells, smooth muscle cells, pericytes, and microglia.