通过两种体外高效策略激活和扩增人脐带血单个核细胞（hUC-MNC）衍生自然杀伤细胞（hUC-NK），以探索生物表型和细胞毒性的相似性和差异性。从健康捐赠者中通过Ficoll基于密度梯度离心法富集脐带血单个核细胞（MNC）。然后，使用“3IL”策略比较了Miltenyi培养基（标记为M-NK）和X-VIVO 15培养基（标记为X-NK）衍生的NK细胞的表型、亚群、细胞存活率和细胞毒性。经过14天的培养，CD3-CD56+ NK细胞的含量从4.25%±0.04%（第0天）升高到分别为71%±0.18%（M-NK）和75.2%±1.1%（X-NK）。与X-NK组相比，M-NK组中CD3+CD4+ T细胞和CD3+CD56+ NKT细胞的比例显著降低。X-NK组中CD16+、NKG2D+、NKp44+、CD25+ NK细胞的百分比高于M-NK组，但X-NK组扩增的NK细胞总数为M-NK组的一半。除了M-NK组中较低比例的Annexin V+凋亡细胞外，在细胞增殖和细胞周期方面，X-NK组和M-NK组之间没有明显差异。在相同的效应细胞比例（E∶T）下，与X-NK组相比，M-NK组中CD107a+ NK细胞的比例更高（P<0.05）。这两种策略都可以在体外高效生成高水平激活的NK细胞，但在生物表型和肿瘤细胞毒性方面存在差异。

To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.