三种原代小胶质细胞纯化培养方法的对比及分析

Correlation and analysis of microglia primary culture methods

目的寻找简捷、高效的原代小胶质细胞纯化培养方法。方法以小胶质细胞原代培养的经典培养方法为基础,通过提高初次接种密度、减少细胞离心过滤、进行营养丧失培养等改进,培养后期分别采用经典机械分离法、低浓度胰酶消化法和利多卡因分离纯化法行细胞分离与纯化,记录形态变化并采用同倍数视野动态计数,借助CD68及OX42抗体间接免疫荧光标记进行鉴定、计算纯度。结果三种分离方法均获得了较高纯度和产量的小胶质细胞,其中利多卡因分离纯化法获得细胞数及纯度明显高于其他分离方法,低浓度胰酶消化法次之。结论小胶质原代细胞培养过程中,利多卡因分离纯化法及低浓度胰酶消化法均可有效提高培养产量及纯度,以利多卡因分离纯化法最佳。

Objective To find an easy and effective method for primary culture and purification of microglia cells. Methods Based on classical microglia primary culture method of McCarthy, improvement like elevation of primary inoculation density, decreace of centrifugal filtration frequency and nutrition deprivation were applied. In the later purification phase of cell culture, three sub-groups such as mechanical separation group, low-concentration enzyme digestion group and lidocaine separation group, were parally operated and cultured. Morphologic changes and cell count under the same magnified field of scope were recorded. Microglia cells were identified and appraised by immunofluorescence mediated by CD68 and OX42 antibodies. Results High puritification quality and quantity of miroglia cells were acquired in all there groups, while the best was lidocaine separation group, followed by low-concentration enzyme digestion group and mechanical separation group. Conclusions During the primary culture of microglia cells,lidocaine separation and low-concentration enzyme digestion can effectively raise the productivity and puritification quality, especially the former.