两步法分离人脐血单个核细胞的最佳条件

背景：如何获得较为纯化、高活性的干细胞,目前未见深入研究报告,也未见一个标准化操作流程方案。 目的：探讨两步法分离人脐血单个核细胞最佳分离条件。 方法：观察羟乙基淀粉在20,30,40,50,60,70 min不同时间沉淀脐血中红细胞的效果;使用人淋巴细胞分离液,分别在800,700,600,500,400 g/min,离心30,25,20 min的条件下分离人脐血单个核细胞。 结果与结论：6%羟乙基淀粉沉淀脐血60 min效果最好;使用密度为(1.077 0±0.000 1) g/mL人淋巴细胞分离液,在4 ℃条件下以700 g/min离心力,离心30 min,洗涤3次,这样获得的人脐血单个核细胞效果最好,所得细胞沉淀中混杂细胞如红细胞及其他细胞碎片较少,人脐血单个核细胞的细胞得率及活力比较高。提示应用羟乙基淀粉沉淀和人淋巴细胞分离液分离两步法,在最佳时间条件下可提高脐血干细胞的回收率。

An exploration of the optimal conditions for isolating umbilical cord blood mononuclear cells by using two-step methods

BACKGROUND:It has not been deeply reported how to obtain high-purity and high-vigor stem cells as well as the standardized operation plan has not been seen, yet. OBJECTIVE:To explore the best condition for isolating human umbilical cord blood mononuclear cells. METHODS:Umbilical cord blood red blood cells precipitated by hydroxyethyl starch were observed in different time points (20, 30, 40, 50, 60, 70 minutes). The human umbilical cord blood mononuclear cells were separated in human lymphocyte separating medium (LSM) by using centrifugation method at 800, 700, 600, 500, 400 g/min for 30, 25, and 20 minutes. RESULTS AND CONCLUSION:The 6% hydroxyethyl starch precipitation for umbilical cord blood red blood cells was best within 60 minutes. Using the human LSM of (1.077 0±0.000 1) g/mL at 4 ℃, centrifugation at 700 g/min for 30 minutes, and rinsing for 3 times the best conditions to harvest human umbilical cord blood mononuclear cells. Under the above-mentioned conditions, not only higher umbilical cord blood mononuclear cells rate and higher mononuclear cells vigor were obtained, but fewer red blood cells and other cell fragments were seen. The best condition for isolating the human umbilical cord blood mononuclear cells using two steps methods (6% hydroxyethyl starch precipitation and LSM) at the optimal time can improve the recovery rate of human umbilical cord blood stem cells.