A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
A protocol is presented for the isolation and primary culture of brain microvascular endothelial cells (BMECs) from neonatal murine brain tissue to generate a robust in vitro model for studying blood-brain barrier function under physiological and pathological conditions.
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.
The blood-brain barrier (BBB) is a highly specialized structure that strictly regulates the exchange of substances between the central nervous system (CNS) and the systemic circulation1. The BBB is primarily formed by brain microvascular endothelial cells connected by tight junctions. These endothelial cells function within a multicellular system known as the neurovascular unit (NVU), which also includes astrocytes, pericytes, neurons, glial cells, and the basement membrane2. Among these components, brain microvascular endothelial cells (BMECs) play a central role by forming a continuous layer with tight junctions, thous....
Access restricted. Please log in or start a trial to view this content.
The Bioethics and Biosafety Committee of the University of Bío-Bío approved this project (Fondecyt grant 1240925). The work with animals was carried out in accordance with the cardinal principles of the three R's in the use of experimental animals13 and in accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health of the United States14. The animals were kept in appropriate environments at the Bioterium of the University of Bío-Bío under standard housing conditions (12 h light/dark cycle, controlled temperature of....
Access restricted. Please log in or start a trial to view this content.
The protocol described here enables the isolation and culture of primary BMECs from neonatal mice (P5). These cells are the main cellular component of the BBB and are therefore a valuable experimental tool for studying cerebrovascular physiology and pathophysiology. Successful isolation and culture of brain microvascular endothelial cells (BMECs) were demonstrated through morphological, phenotypic, and functional assessments.
The isolation workflow resulted in a cell suspension derived from di.......
Access restricted. Please log in or start a trial to view this content.
The isolation of BMECs is an instrumental tool for studying the mechanisms that regulate the physiology of the BBB and its dysfunction in various central nervous system (CNS) pathologies4,9, including stroke. Unlike immortalized cell lines, primary BMEC cultures preserve key phenotypic and functional features of the brain endothelium, including the expression of tightly bound proteins, and an angiogenic capacity representative of the in vivo. context
Access restricted. Please log in or start a trial to view this content.
The authors have no conflicts of interest to declare.
Portions of this manuscript were written with OpenAI's ChatGPT (GPT-4, July 2025 version) to improve clarity, language, and coherence. The authors critically reviewed, edited, and approved all AI-assisted content. No AI tools were used for data analysis, result interpretation, or the generation of scientific conclusions.
The authors would like to thank the researchers belonging to GRIVAS Health and NEUROVAS for their valuable input. This study was funded by Fondecyt Regular 1240295.
....Access restricted. Please log in or start a trial to view this content.
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 60 mm culture plates | Corning | 430166 | |
| 96 Well culture plates | Corning | 41122107 | |
| Acetic acid | Sigma-Aldrich | A6283 | |
| Antibiotic-Antimycotic (optional) | Gibco | 15240062 | |
| Anti-CD31 | Santa Cruz | sc-46694 | 1:100 |
| Anti-GFAP | Thermo Fisher Scientific | 13-0300 | 1:500 |
| Anti-GLUT1 | Abcam | ab115730 | 1:200 |
| Anti-Iba1 | Santa Cruz | sc-32725 | 1:500 |
| Anti-mouse IgG Alexa Fluor (594 Dye) | Thermo Fisher Scientific | A-11005 | 1:500 |
| Anti-rabbit IgG Alexa Fluor (488 Dye) | Thermo Fisher Scientific | A-11008 | 1:500 |
| Automatic and serological pipettes | Oxford LP | OXL.OXL.OSP-5C | |
| Basal medium (for collagen solution) | Gibco (DMEM) | 11965092 | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A9647 | |
| Collagenase type II | Sigma-Aldrich | C6885 | |
| DAPI | Thermo Fisher Scientific | 62248 | 1 µg/mL |
| DMEM (high glucose) | Thermo Fisher Scientific | 11965092 | |
| EndoGRO-MV Complete Medium | Merck Millipore | SCME004 | Endothelial cell growth medium |
| Falcon tubes (15 & 50 mL) | Corning® | 352096 / 352070 | |
| Fetal Bovine Serum (FBS) | Gibco (Thermo Fisher) | 16000044 | |
| Fluorescence Microscope | Motic | BA410 | |
| Gloves, masks, lab consumables | Various suppliers | — | |
| Heparin (100 U/mL) | Sigma-Aldrich | H3393 | |
| Inverted Microscope | Olympus | CKX41 | |
| Magnetic separation magnet | EasySep | NC9284020 | |
| Orbital Shaker (37 °C) | Thermo Fisher Scientific | SK-0180-S | |
| Parafilm | Bemis | PM996 | |
| PBS (1×) | Thermo Fisher Scientific | 10010023 | |
| Refrigerated Centrifuge | HITACHI | CT5RE | |
| Scalpel | Fine Science Tools | Various | |
| Scissors and fine tweezers | Fine Science Tools | Various | |
| Sterile nitrocellulose membrane | BIO-RAD | 1620094 | |
| Type I collagen | Corning | 30394 |
Access restricted. Please log in or start a trial to view this content.
This article has been published
Video Coming Soon