Isolation And Culture of Rat Intestinal Mucosal Microvascular Endothelial Cells Using Immunomagnetic Beads And Their Marker Expression

Abstract Microvascular endothelial cells (MVECs) have been an important tool in many research fields, while their purification remained challenging. This study aimed to establish a method for isolating rat intestinal mucosal MVECs using immunomagnetic beads, and characterize their proliferation ability and marker expression. Rat jejunum mucosas were scraped and digested by collagenase type II to obtain primary cell culture by the differential adhesion method. Magnetic beads were coated with anti-CD31 antibodies and incubated with the single-cell suspension of the primary cell culture. The CD31+ cells were separated using an automatic magnetic separation system, and their proliferation ability were assayed at different passages. The expression of factor VIII (FVIII), CD31, and CD34 was detected by immunofluorescence staining. Results indicated that rat intestinal mucosal MVECs grew into a contact-inhibited cobblestone-like monolayer after about 6 days, whose proliferation ability had no significant decline at passage 20. Three markers were generally expressed in the cells, and the fluorescence intensity of FVIII was higher after magnetic separation than before, and those of CD31 and CD34 remained similar. In conclusion, highly pure MVECs can be isolated from the primary cell culture of rat intestinal mucosas using magnetic beads coated with anti-CD31 antibodies, and magnetic separation may influence the expression of FVIII.

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Primary cell culture of canine corneal endothelial cells

Veterinary Ophthalmology ◽

10.1111/vop.12924 ◽

2021 ◽

Author(s):

Kazuki Tajima ◽

Misaki Okada ◽

Rina Kudo ◽

Yuya Otaka ◽

Mizuki Kita ◽

...

Keyword(s):

Cell Culture ◽

Endothelial Cells ◽

Primary Cell Culture ◽

Primary Cell ◽

Corneal Endothelial Cells

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Multimodal characterization of the murine neurovascular niche using a novel microvascular isolation protocol

10.1101/2021.08.31.458349 ◽

2021 ◽

Author(s):

Katrine Dahl Bjørnholm ◽

Michael Vanlandewijck ◽

Francesca Del Gaudio ◽

Urban Lendahl ◽

Per Nilsson ◽

...

Keyword(s):

Cell Culture ◽

Endothelial Cells ◽

Single Cell ◽

Primary Cell Culture ◽

Cell Types ◽

Primary Cell ◽

Fast Method ◽

Vascular Cells ◽

Vascular Component ◽

The Brain

AbstractThe blood-brain barrier (BBB) is central to separate blood from the extracellular fluids of the brain. To understand disease-related changes in the BBB is pivotal and such changes can increasingly be studied by single-cell RNA sequencing (scRNAseq), which provides high-resolution insight into gene expression changes related to the pathophysiological response of the vasculature. However, analysis of the vascular cells in the brain is challenging due to the low abundance of these cells relative to neuronal and glial cells, and improved techniques for enrichment of the vascular component is therefore warranted. The present study describes a method whereby panning with CD31-coated magnetic beads allows isolation of brain vasculature without the need for transgenic reporter lines or FACS sorting. The protocol was tested in three modalities: isolation of cells for scRNAseq, western blot (WB) analysis, and primary cell culture. For scRNAseq, a total of 22,515 single-cell transcriptomes were generated from 12-months old mice and separated into 23 clusters corresponding to all known vascular and perivascular cell types. The most abundant cell type was endothelial cells (EC) (Pecam1- and Cdh5-positive), which dispersed into clusters of arterial, capillary, and venous EC according to previously established BBB arterio-venous zonation markers. Furthermore, we identified clusters of microglia (Aif1-positive), one cluster of fenestrated endothelial cells (Plvap-positive; Cldn5-negative), a cluster of pericytes (Kcnj8- and Abcc9-positive) and a cluster of vascular smooth muscle cells (VSMC) (Acta2- and Tagln-positive). WB analysis using established markers for the different cell types (CD31 (EC), SM22 (VSMC), PDGFRB (pericytes), GFAP (astrocytes), and IBA1 (microglia) confirmed their presence in the isolated vascular component and suggests that the protocol is suitable for future proteomic analysis. Finally, we adapted the isolation protocol to accommodate primary culture of brain vascular cells. In conclusion, we have successfully established a simple and fast method for isolating microvasculature from the murine brain independent of cell sorting and alleviating the need to use reporter mouse lines. The protocol is suitable for a multitude of testing modalities, including single-cell analyses, WB and primary cell culture.

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