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

IF 2.3 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-08-15 DOI:10.1016/j.jneumeth.2025.110558

Yuanyuan Wang , He Jiang , Junyan Yan , Shijie Li , Zihao Xin , Jiaxiong He , Sihan Wang , Caixia Fan , Lulu Zhang

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引用次数: 0

Abstract

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.

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人脊髓小胶质细胞/巨噬细胞培养：精确酶消化和非酶纯化

脊髓损伤（SCI）和神经系统疾病是重大的医学挑战，小胶质细胞/巨噬细胞对神经炎症和修复至关重要。使用动物或人类大脑小胶质细胞/巨噬细胞的传统体外模型存在物种/区域差异，限制了翻译。缺乏有效的人脊髓小胶质细胞/巨噬细胞（hSCM）分离方法阻碍了脊髓损伤机制研究和药物筛选。本研究通过两个关键创新优化了hSCM分离/培养方案：Accutase消化：机械切碎+ 37°C Accutase 15 min取代传统的机械解离，提高单细胞产量（>； 95% %存活率），同时保留表面抗原（例如，Iba-1, CD45）。两步非酶净化：利用小胶质细胞/巨噬细胞和星形胶质细胞之间的粘附力差异，“适度扩张+握手”去除非粘附细胞，避免酶损伤并保持>； 90 %的活力。结果细胞特征：分离的hSCM表现出典型的静息状态形态（杆状/支状过程），免疫荧光和流式细胞术检测结果显示小胶质细胞/巨噬细胞标志物（Iba-1 + /DAPI + >95 %,CD45 94.18 %,CD11b 80.9 %）。纯度和活力：纯度>； 90 %，纯化后活力>； 92 %。细胞保持了增殖能力（倍增时间48-72 h）和表型稳定性（Iba-1 +在3代中+ 90 %）。效率更高：单细胞产率（95 %）超过传统的机械分离（~ 60-70 %）。高纯度：非酶纯化达到>； 95 %纯度，优于经典的小鼠脑小胶质细胞/巨噬细胞方法（85-90 %）。温和的解离：与苛刻的胰蛋白酶相比，精确酶保持抗原的完整性。结论该系统建立了标准化、高纯度的hSCM模型，填补了人类特异性SCI研究的关键空白。它促进了小胶质细胞/巨噬细胞免疫调节机制、药物筛选和跨物种翻译的研究。未来的应用可能会整合诱导iPSC技术进行个性化疾病建模，以推进脊髓损伤的精准医疗。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.