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Updated: Jul 20, 2026
Culturing Microglia from the Neonatal and Adult Central Nervous System
Published on: August 9, 2013
Human spinal cord microglia/macrophages culture: Accutase digestion and non-enzymatic purification
Yuanyuan Wang1, He Jiang2, Junyan Yan1
1School of Life and Environmental Sciences, Shaoxing University, Shaoxing, Zhejiang 312000, China.
Background:
Spinal cord injury (SCI) and neurological diseases pose major medical challenges, with microglia/macrophages critical for neuroinflammation and repair. Traditional in vitro models using animal or human brain microglia/macrophages suffer from species/regional differences, limiting translation. The lack of efficient isolation methods for human spinal cord microglia/macrophages (hSCM) has hindered SCI mechanistic research and drug screening.
New Method:
This study optimized an hSCM isolation/culture protocol with two key innovations: Accutase digestion: Mechanical mincing+ 37 °C Accutase for 15 min replaces traditional mechanical dissociation, enhancing single-cell yield (>95 % viability) while preserving surface antigens (e.g., Iba-1, CD45). Two-step non-enzymatic purification: Using adhesion force differences between microglia/macrophages and astrocytes, "moderate expansion+hand-shaking" removes non-adherent cells, avoiding enzymatic damage and maintaining> 90 % viability.
Results:
Cell characteristics: Isolated hSCM showed typical resting-state morphology (rod-shaped/branched processes) and expressed microglia/macrophages markers (Iba-1⁺/DAPI⁺ >95 %, CD45 94.18 %, CD11b 80.9 %) via immunofluorescence and flow cytometry. Purity and viability: Purity > 90 %, viability > 92 % post-purification. Cells retained proliferative capacity (doubling time 48-72 h) and phenotypic stability (Iba-1⁺ >90 % over 3 passages).
Comparison With Existing Methods:
Higher efficiency: Single-cell yield (95 %) exceeds traditional mechanical dissociation (∼60-70 %). Superior purity: Non-enzymatic purification achieves > 95 % purity, outperforming classical mouse brain microglia/macrophages methods (85-90 %). Gentler dissociation: Accutase preserves antigen integrity versus harsh trypsin-based protocols.
Conclusions:
The system establishes a standardized, high-purity hSCM model, filling critical gaps in human-specific SCI research. It facilitates studies on microglia/macrophage immunoregulatory mechanisms, drug screening, and cross-species translation. Future applications may integrate induced iPSC technology for personalized disease modeling to advance precision medicine in SCI.
Insights
This study presents a novel method for isolating human spinal cord microglia/macrophages (hSCM), crucial for understanding spinal cord injury (SCI) and neurological diseases. The optimized protocol yields high-purity, viable cells for advanced research and drug screening.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Spinal cord injury (SCI) and neurological diseases present significant challenges, with microglia/macrophages playing a key role in neuroinflammation and repair.
- Existing in vitro models using animal or human brain cells have limitations due to species and regional differences.
- A lack of efficient isolation methods for human spinal cord microglia/macrophages (hSCM) has hindered SCI research and drug development.
Purpose of the Study:
- To develop and optimize a protocol for the efficient isolation and culture of high-purity human spinal cord microglia/macrophages (hSCM).
- To establish a reliable in vitro model for studying human-specific neuroinflammation and repair mechanisms in SCI.
- To facilitate drug screening and improve the translational relevance of SCI research.
Main Methods:
- Optimized hSCM isolation using Accutase digestion for enhanced single-cell yield (>95% viability) and preservation of surface antigens.
- Implemented a two-step non-enzymatic purification method based on cell adhesion properties to achieve high purity (>90%) while maintaining cell viability (>90%).
- Characterized isolated hSCM for morphology, marker expression (Iba-1, CD45, CD11b), purity, viability, proliferative capacity, and phenotypic stability over passages.
Main Results:
- The optimized protocol achieved a single-cell yield of >95% with >92% viability, surpassing traditional mechanical dissociation methods.
- Isolated hSCM exhibited high purity (>90%) and expressed characteristic microglia/macrophage markers (Iba-1⁺/DAPI⁺ >95%, CD45 94.18%, CD11b 80.9%).
- The cells demonstrated stable proliferative capacity (doubling time 48-72h) and maintained phenotypic stability over three passages.
Conclusions:
- A standardized, high-purity hSCM model has been established, addressing critical gaps in human-specific SCI research.
- This model will facilitate studies on microglia/macrophage immunoregulatory mechanisms, enable effective drug screening, and improve cross-species translation.
- Future applications include integrating induced pluripotent stem cell (iPSC) technology for personalized disease modeling in SCI, advancing precision medicine.
