Isolation and culture of rat intestinal mucosal microvascular endothelial cells using immunomagnetic beads

• Published in: Journal of immunological methods 2022-08, Vol.507, p.113296-113296, Article 113296
• Main Authors: Wu, Yanmei, Wang, Zhaoli, Hu, Ge, Zhang, Tao
• Format: Article
• Language: English
• Subjects: CD31; CD34; Factor VIII; Intestinal mucosa; Microvascular endothelial cells
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2022.113296

Microvascular endothelial cells (MVECs) have been an important tool in many research fields, while their purification remained challenging, particularly from thin and fragile tissues, such as murine intestinal mucosae. Therefore, we established an immunomagnetic method for isolating rat intestinal mucosal MVECs using an automatic magnetic separation system from the primary cell culture, which was preliminarily purified by the differential adhesion combined with mechanically scraping method. The CD31+ cells were separated and their purity was about 96.5%, which grew into a contact-inhibited cobblestone-like monolayer after about 6-day incubation. Transmission electron microscopy analysis showed the presence of Weibel-Palade bodies. The endothelial tubes formed on Matrigel for about 4-h cultivation. The MVECs could grow well until at least passage 10. Immunofluorescence staining indicated that factor VIII (FVIII), CD31 and CD34 were generally expressed in the MVECs. The fluorescence intensity of FVIII was higher after magnetic separation than before, while those of CD31 and CD34 didn't have significant difference. In conclusion, highly pure MVECs were isolated from rat intestinal mucosae using magnetic beads coated with anti-CD31 antibodies, and magnetic separation may influence the expression of FVIII. •The separating method of highly pure MVECs based on an automatic immunomagnetic separation system from preliminarily purified primary cell cultures of rat intestinal mucosae was established.•Rat intestinal mucosal MVECs were characterized by transmission electron microscopy analysis, the tube formation assay, the proliferation ability assay, and the endothelial marker expression.•Effects of magnetic separation on the endothelial marker expression were paid attention to.•The suggestions for operating the automatic magnetic separation system and choosing the antibody suitable for the immunomagnetic separation were provided.