人脾脏巨噬细胞的分离与纯化

Isolation and purification of macrophages from human spleen

目的 探索分离纯化大量人脾脏巨噬细胞 (MΦ)的方法。方法 选取 10例手术切除的脾脏 ,机械研磨法获得脾脏组织细胞混悬液。采用贴壁培养的方法进行MΦ的分离与纯化 ,相差显微镜下动态观察 ,明确贴壁培养的合适时间 ,详细记录MΦ分离纯化过程各步骤所需时间。收获的MΦ ,台盼蓝染色判定活力并计数 ,统计出平均每克脾脏组织收获的MΦ数及其纯度。相差显微镜、透射电镜观察分离纯化MΦ的形态特征。结果 分析贴壁培养各时间段的细胞纯度及活力 ,确定MΦ培养的合适时间为 2～ 3h。贴壁培养纯化后 ,平均从每克脾脏组织可收获 (0 .2 5± 0 .0 3)× 10 8个MΦ ,纯度为 (90± 5 ) % ,细胞活力≥ 99% ,细胞结构及各种细胞器结构均完整。结论 贴壁培养方法分离纯化人脾脏MΦ是成功可行的 ,收获的MΦ数量大、纯度高、活力好 。

Objective To develop a novel me th od of isolating and purifying a great number of macrophages from human spleen. Methods Ten cases of excisional human spleen were selected. In order to obtain a spleen cell suspension, human spleens were cut into small pieces, mechanically ground in culture medium of RPMI-1640 and filtered. Macrop hages were isolated and purified by anchoring cultivation, and were observed con secutively under phase-contrast microscope to identify the optimal time of cult ivation. Meanwhile, the time needed during each procedure was recorded in detail . The viability of purified macrophages was assessed with trypan blue exclusion, and the average yield per gram of spleen and purity of macrophages were calcula ted. Morphological characteristics of isolated macrophages were detected by phas e-contrast microscope and transmission electron microscope. Results The optimal time of anchoring cultivation was confi rmed to be 2 ～3 hours. The average yield of macrophages isolated freshly was (0 .25±0.03)×10 8 per gram of spleen, purity was 90%±5%, and the viability was constantly ≥99%. Cell structure and organelle ultrastructure of macrophages were intact. Conclusion A great number of macrophages with high degree o f purity and viability can be successfully isolated and purified from human sple en by anchoring cultivation. This novel method of isolation and purification wil l undoubtedly facilitate the functional investigation of macrophages in human sp leen.