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Updated: Dec 18, 2025
Isolation of Primary Murine Brain Microvascular Endothelial Cells
Published on: November 14, 2014
[Primary culture and identification of mouse brain microvascular endothelial cells]
Zhaode Liu1, Huagen Ma2, Yuanyu Tang3
1College of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China.
Abstract:
Objective To develop and establish a simple and repeatable method of primary culture of mouse brain microvascular endothelial cells (BMECs) of relatively high purity. Methods Isolated and cultured from the brain tissue of ICR mice aged 2-3 weeks old, the mouse BMECs were gained through physical fragmentation methods such as cutting up and passing through cell sieve, BSA density gradient centrifugation and chemical enzyme digestion. The digestion time of 1 g/L type II collagenase was strictly controlled within 15-20 minutes. The cultured cells were identified by cell morphological observation and immunocytochemical staining to detect factor VII-associated antigen. Results In 24 hours after seeding, the spindle-shaped or polygon-shaped cells began to migrate from the micro vessel segment and gradually gathered and grew in clusters; after 7 days, the fused cells showed a typical single layer and arranged in the shape of paving stone. VII-associated antigen showed positive in the cytoplasm which was brown red. The purity of microvascular endothelial cells was above 95%. Conclusion We have successfully established a simple and repeatable primary culture method for mouse BMECs of relatively high purity.
Insights
Researchers developed a simple method to culture high-purity mouse brain microvascular endothelial cells (BMECs). This technique is repeatable and suitable for scientific research, yielding over 95% pure BMECs.
Area of Science:
- Neuroscience
- Cell Biology
- Biotechnology
Background:
- Brain microvascular endothelial cells (BMECs) are crucial for the blood-brain barrier.
- Establishing pure BMEC cultures is vital for studying neurovascular diseases.
- Existing methods for BMEC isolation can be complex and yield low purity.
Purpose of the Study:
- To develop and validate a simple, repeatable primary culture method for mouse BMECs.
- To achieve high purity of isolated BMECs for research applications.
- To optimize isolation and culture conditions for efficient BMEC expansion.
Main Methods:
- Primary culture of BMECs from 2-3 week old ICR mouse brains.
- Utilized physical fragmentation, BSA density gradient centrifugation, and enzymatic digestion (Type II collagenase, 15-20 min).
- Identified cells via morphology and immunocytochemistry for Factor VII-associated antigen.
Main Results:
- Cultured cells exhibited spindle or polygonal shapes, migrating and forming clusters within 24 hours.
- After 7 days, cells formed a monolayer with a cobblestone-like appearance.
- Immunocytochemistry confirmed positive Factor VII-associated antigen in the cytoplasm (brown-red).
- Achieved a purity of mouse BMECs exceeding 95%.
Conclusions:
- Successfully established a simple and repeatable primary culture method for mouse BMECs.
- The developed method yields high-purity BMECs, suitable for further research.
- This technique provides a reliable source of BMECs for studying blood-brain barrier functions and related pathologies.
