Human spinal cord microglia/macrophages culture: Accutase digestion and non-enzymatic purification.

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.