Isolation and Culture of Mouse Brain Microvascular Endothelial Cells.

Brain microvascular endothelial cells (BMECs) are the main cellular component of the blood-brain barrier (BBB) and play a key role in regulating molecular transport and maintaining central nervous system homeostasis. Reliable in vitro models of brain endothelium are essential for studying BBB physiology and dysfunction in neurological and systemic diseases. A reproducible protocol for the isolation and primary culture of BMECs from neonatal murine cerebral cortex is described. The method includes cortical tissue dissection, mechanical and enzymatic dissociation, removal of myelin debris using a bovine serum albumin (BSA) density gradient, and enrichment of endothelial cells using CD31-based immunoselection. Isolated cells are subsequently cultured on collagen-coated plates and characterized by morphological assessment, immunofluorescence detection of endothelial markers, angiogenic tube formation assays, and transendothelial electrical resistance (TEER) measurements to evaluate barrier properties. This protocol provides a reliable approach for obtaining primary BMEC cultures suitable for investigating BBB biology, endothelial signaling, and neurovascular interactions under physiological and pathological conditions.