二甲基亚砜与羟乙基淀粉程控降温与低温冰箱降温保存造血干细胞的比较

Cryopreservation of Hemotopoietic Stem Cells Using Dimethyl Suflfoxide and Hydroxyethyl Starch in Low Temperature Refrigerator and Autocontrolled Programmed Cryogenic

目的为造血干细胞的低温保存选择合适的保护剂和简便的保存方法。方法血细胞分离机分离的外周血细胞,加入二甲基亚砜或羟乙基淀粉,超低温冰箱降温(保存)或自控程序降温液氮保存,定期检测细胞的粒单系造血细胞、长期培养起始细胞、CD34+细胞、台盼蓝拒染细胞,计回收率。结果超低温冰箱降温保存与自控程序降温在液氮内保存,复温后粒单系造血细胞、长期培养起始细胞、CD34+细胞、台盼蓝拒染细胞的回收率,就降温与保存条件而言,两者差异不明显,但就保护剂来说5%二甲基亚砜-6%羟乙基淀粉优于10%二甲基亚砜。超低温冰箱降温液氮保存和超低温冰箱降温保存,与自控程序降温液氮保存比较,在1年内保存良好,无明显差异(P>0.05);但2年后,超低温冰箱降温保存者细胞活力下降。细胞复温后,细胞内保护剂不稀释也不洗涤,在室温放置,则对细胞活力有影响,以10%二甲基亚砜为保护剂影响最大;而5%二甲基亚砜-6%羟乙基淀粉为保护剂者虽然对细胞也有影响,但比10%二甲基亚砜为保护剂者影响要小。结论保存外周血干细胞时,我们推荐使用5%二甲基亚砜-6%羟乙基淀粉为保护剂;保存期在1年内,可用超低温冰箱降温保存;超过1年时,超低温冰箱降温用液氮保存。细胞复温后,细胞内保护剂应立即稀释或去除。

Objective To explore proper preservatives and simple conditions for cryoprereservation of hemotopoietic stem cells. Methods Bone marrow cells and peripheral blood mononucleated cells obtained by blood cell seperator were added to 5% dimethyl suflfoxide （DMSO） and 6% hydroxyethyl starch （HES, 5%DMSO-6%HES）, then preserved in low temperature refrigerator. Granulocyte-macrophage colony forming unit （GM-CFU）, long term culture initiating cells（I.TC-IC）, CD34^＋ cells and typhan blue resistance cells（TBRC） were counted compared with the cells preserved in 10% DMSO by autocontrolled programmed cryogenic system in liquid nitrogen. Results CFU-GM,LTC-IC,CD34^＋ cells and TBRC showed no significant difference between low temperature refrigerator group and liquid nitrogen group. These parameters were not significantly different within one year when the cells were preserved in liquid nitrogen after laid in low tempreture refrigerator, or directly in low tempreture refrigerator, or in liquid nitrogen with autocontrolled programmed cryogenic system,however the cell recovery rate decreased after half a year when the cells were directly preserved in low tempreture refrigerator. The cell activity declined after recovery at room temperature if the preservatives were not removed or diluted in both groups, the group of 10%DMSO was more affected than 5%DMSO-6%HES group was. Conclusion 5%DM- SO-6%HES is better than 10%DMSO as preservative for hemopoietic stem cells. The cells in the three groups above will be preserved well within one year. If the cells will be preserved for long duration,liquid nitrogen is the best choice. The preservatives should be diluted immediately after the cells are recovered.