目的:探讨脐血单个核细胞的提取、冻存及复苏工艺,实现脐血单个核细胞的长久保存。方法:采集足月妊娠者脐血40~180ml,运用Ficoll密度梯度离心法和AXP脐带血自动分离系统与Ficoll密度梯度离心法两步法分离脐血单个核细胞,采用非程控降温法降温,将细胞保存于-196℃液氮中。比较分析不同分离方法分离人脐血单个核细胞的细胞得率及活力;冷冻保护液配制分2组:无血清培养液+10%DMSO+10%右旋糖酐和自体血浆+10%DMSO+10%右旋糖酐,根据冻存时间进行细胞复苏,分析其冻存前后的细胞活力、单个核细胞回收率、CD34+细胞数和CFU形成能力。结果:Ficoll密度梯度离心后获得的脐血单个核细胞活力为99.70%±0.30%,回收率为54.40%±4.52%;两步法分离脐血单个核细胞活力为99.60%±0.40%,回收率为62.12%±3.78%,2组回收率差异有统计学意义(P<0.05)。冻存6、12、24个月后,各时间点单个核细胞无血清培养液组复苏回收率和活力分别为80.75%±4.60%和91.75%±5.42%;自体血浆组复苏回收率和活力分别为81.55%±4.50%和92.65%±4.92%,组间差异无统计学意义。复苏后CD34+细胞占1.25%±0.70%,集落形成总数为(32.10±29.60)/105。结论:两步法分离脐血单个核细胞的回收率高于一步法,两种保护液的保护效果无显著差异。 OBJECTIVE: To investigate the extraction, cryopreservation and recovery of umbilical cord blood mononuclear cells for long-term preservation of umbilical cord blood mononuclear cells. Methods: Umbilical cord blood 40 ~ 180ml was collected from full-term pregnancy. Umbilical cord blood mononuclear cells were isolated by Ficoll density gradient centrifugation, AXP cord blood automated system and Ficoll density gradient centrifugation. Cells are stored in liquid nitrogen at -196 ° C. The cell viability and viability of human umbilical cord blood mononuclear cells isolated by different isolation methods were comparatively analyzed. The cryoprotectants were divided into 2 groups: serum-free medium + 10% DMSO + 10% dextran and autologous plasma + 10% DMSO + 10% dextran , Cell resuscitation was performed according to cryopreservation time, cell viability, mononuclear cell recovery rate, CD34 + cell number and CFU formation ability before and after cryopreservation were analyzed. Results: The viability of cord blood mononuclear cells obtained by Ficoll density gradient centrifugation was 99.70% ± 0.30% and the recovery was 54.40% ± 4.52%. The activity of cord blood mononuclear cells was 99.60% ± 0.40% (62.12% ± 3.78%). There was significant difference between the two groups (P <0.05). After 6, 12 and 24 months of cryopreservation, the recovery rate and viability of mononuclear cells in serum-free medium at each time point were 80.75% ± 4.60% and 91.75% ± 5.42%, respectively. The recoveries and viability of autologous plasma were 81.55% ± 4.50% and 92.65% ± 4.92% respectively. There was no significant difference between the two groups. After recovery, CD34 + cells accounted for 1.25% ± 0.70%, and the total number of colony formation was (32.10 ± 29.60) / 105. Conclusion: The recovery rate of cord blood mononuclear cells by two-step method is higher than that of one-step method. There is no significant difference between the two protective solutions.