Neurosurgery Institute, Key Laboratory on Brain Function Repair and Regeneration of Guangdong, Zhujiang Hospital, Southern Medical University, 510282, Guangzhou, China.
Cell Mol Neurobiol. 2012 Jan;32(1):67-75. doi: 10.1007/s10571-011-9735-9. Epub 2011 Jul 16.
Microglia, implicating in such neuro-pathologies as brain inflammation, neurodegeneration, glioma, and neurogenesis, play an important role in central nervous system. Advanced research on microglia is crucial in exploring the neuro-pathology and neuro-physiology of these diseases, so how to culture large numbers of microglia in vitro becomes the base of a research. The wildly used method, at present, obtaining microglia from murine cannot fulfill the requirement of research, costing too much time and needing too many rats. We intend to introduce an optimized method that can harvest large quantities of microglia with high purity. Neonatal 2-3 days old Wistar rats were sacrificed and the cerebral cortices were trypsinized. We primarily cultured mixed cortical cells for 8-10 days. The microglia were harvested from the liquid supernatant; the left cells in the mixed cortical glial culture were passaged at a 1:2 density. After another 8-10 days of culture, microglia were collected again. And then, we passaged the left cells again for acquiring microglia from the third collection. We did not add additional mitogens in the experiment. At last, on average, 7.0 × 10(6) microglia were collected from one neonatal rat. By this modified method, much more microglia can be effectively and easily harvested comparing with the usual protocol before. We compared the characteristics of microglia harvested from these three passages, such as morphology, phenotype, purity, and abilities on proliferation, secretion, and phagocytosis. The cells presented typical microglia morphology, having phenotype markers of CD11b/c and CD45. The microglia from these three passages retained similar phagocytosis and secretion functions. Expanded population of microglia for investigation can be provided by this easy method in a short time with little cost and few rats.
小胶质细胞在中枢神经系统中发挥重要作用，涉及脑炎症、神经退行性变、神经胶质瘤和神经发生等神经病理学。深入研究小胶质细胞对于探索这些疾病的神经病理学和神经生理学至关重要，因此如何在体外培养大量小胶质细胞成为研究的基础。目前，从鼠脑中获得小胶质细胞的广泛应用方法既耗时又费力，需要大量的老鼠。我们旨在引入一种优化的方法，可以高效地收获高纯度的大量小胶质细胞。将新生 2-3 天的 Wistar 大鼠处死，分离大脑皮质，用胰蛋白酶消化。我们首先培养混合皮质细胞 8-10 天。将小胶质细胞从液体上清液中收获；将混合皮质胶质培养物中剩余的细胞以 1:2 的密度传代。再培养 8-10 天后，再次收集小胶质细胞。然后，我们再次传代剩余的细胞，从第三次收集物中获取小胶质细胞。实验中未添加其他有丝分裂原。最后，从一只新生大鼠中平均可收集到 7.0×10(6)个小胶质细胞。通过这种改良方法，与之前的常规方案相比，可以更有效地从新生大鼠中收获更多的小胶质细胞。我们比较了从这三个传代中收获的小胶质细胞的特征，如形态、表型、纯度以及增殖、分泌和吞噬能力。这些细胞呈现出典型的小胶质细胞形态，具有 CD11b/c 和 CD45 等表型标志物。这三个传代的小胶质细胞保留了相似的吞噬和分泌功能。这种简单的方法可以在短时间内以较低的成本和较少的老鼠提供用于研究的小胶质细胞扩增群体。
