Isolation and purification of macrophages from human spleen

Objective: To develop a novel method 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. Macrophages were isolated and purified by anchoring cultivation, and were observed consecutively under phase-contrast microscope to identify the optimal time of cultivation. 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 calculated. Morphological characteristics of isolated macrophages were detected by phase-contrast microscope and transmission electron microscope. Results: The optimal time of anchoring cultivation was confirmed to be 2-3 hours. The average yield of macrophages isolated freshly was (0.25±0.03) × 10⁸ 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 of purity and viability can be successfully isolated and purified from human spleen by anchoring cultivation. This novel method of isolation and purification will undoubtedly facilitate the functional investigation of macrophages in human spleen.