Novel Strategy for Isolation of Mice Bone Marrow –Derived Endothelial Cells (BMECs)
Background: In the bone marrow microenvironment, endothelial cells (ECs) are individual cells that form part of the sinusoidal blood vessels called "bone marrow endothelial niche." They account for less than two percent of the bone marrow cells. They may play critical functions in generating growth and inhibitory factors that promote the recovery of the hematopoietic stem cells. Methods: Two steps approach for isolation of bone marrow endothelial cells from mice. In brief, the bone marrow
... e bone marrow extracted from the mice long bones and culture overnight with DMEM supplemented with 20% FBS and antibiotics. The floating cells discarded, and the adhered section detaches with accutase and bone marrow endothelial cells selected using CD31 microbeads. The isolated bone marrow endothelial cells were cultured in dish pre-coated with rat-tail collagen type 1 with endothelial growth factor media. The cells were verified by confocal microscopy for morphology and tube formation by matrigel assay. We validate the purity of the cells by flow cytometry, RT-qPCR, immunofluorescence staining, and immunoblotting. Finally, we induced the bone marrow endothelial cells with recombinant tumor necrosis factor-alpha.Results: Our findings prove that the cell isolated are characteristic of bone marrow endothelial cells and response to tumor necrosis factor-alpha by an increase in proliferation and enhance the expression of adhesion molecules.Conclusions: This method simplifies the extraction of primary bone marrow endothelial cells for in vitro studies. The significance of this method will provide an excellent opportunity for stem cell research of endothelial cells function and dysregulation in vitro studies of mice model.