Optimized protocols for the simultaneous isolation of primary brain microvascular endothelial cells and primary neurons with high purity and functional maturation from individual newborn mice

Abstract

Background

Current neurovascular unit isolation requires processing brain microvascular endothelial cells (BMECs) and neurons from separate animals, preventing concurrent analysis of neurovascular crosstalk within identical genetic/physiological contexts.

New methods

We developed an enzymatic digestion/bovine serum albumin density gradient technique that enabled the simultaneous isolation of neural tissue and microvascular segments from individual mice. The neural tissue was filtered and centrifuged for primary cortical neuron culture on poly-L-lysine-coated plates. Microvascular segments were subjected to collagenase/dispase digestion and Percoll gradient centrifugation for BMEC culture on fibronectin-coated plates. Cellular purity was quantified via immunofluorescence, and BMEC functionality was assessed by tight junction expression, transendothelial electrical resistance (TEER), tubulogenesis, and secretory function. Neuronal characteristics were evaluated using morphometric analysis, detection of neurotransmitter secretion, and sensitivity to oxygen-glucose deprivation (OGD).

Results

High-purity BMECs and primary cortical neurons were successfully isolated by enzymatic digestion combined with density-gradient centrifugation. Primary BMECs exhibited fibronectin-dependent adhesion during initial plating, with a significantly enhanced adhesive capacity observed in passages 2 and 3. Tubulogenesis assays demonstrated superior tube-forming capacity of primary BMECs compared b.End3 cells. TEER and nitric oxide (NO) secretion decreased by 38.31 % and 26.1 %, respectively, following OGD. Primary cortical neurons displayed a characteristic somatic morphology with extensive neurite arborization and heightened sensitivity to OGD. The GABA level in the OGD group was 2.01 times higher than that in the control group and decreased by 52.5 % after reoxygenation.

Comparison with existing methods

Unlike conventional multi-animal protocols that introduce inter-individual variability, our single-mouse approach eliminates genetic confounders while reducing processing time by 40–60 % and yielding higher purity. Furthermore, primary BMECs and neurons maintained their original characteristics, including morphology, angiogenic capacity, and secretory function.

Conclusion

This novel platform reliably co-isolated functional primary BMECs and cortical neurons from individual mice, providing unprecedented fidelity for modeling neurovascular interactions in disease contexts.