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用人类脐血干/祖细胞代替骨髓移植,治疗辐射损伤性疾病,并为核事故受害者的救治建立脐血干细胞库是一重要课题,而脐血造血干细胞的分离与冻存是建立脐血干细胞库、进行干细胞移植的关键。研究了脐血造血干细胞的分离和冻存方法,首先用密度梯度离心法以不同比重的淋巴细胞分离液(Ficoll液)分离脐血单个核细胞(mononuclear cells,MNC),计数,并用CD_(34)单抗经流式细胞仪进行表型分析;然后加冻存保护剂梯度降温,冻存于液氮中,再间隔一定时间常规复苏MNC。采用“三种梯度降温法”、“五种不同细胞浓度”和“两种细胞冻存保护剂”比较了不同条件下脐血MNC的分离和冻存效果。结果显示,使用比重为1.064的Ficoll液较常规1.077所分离的MNC中CD_(34)~+细胞百分数高,前者约为后者的3倍,但细胞总数较少;梯度降温以直接放入液氮瓶口内(液相之上的气相中)过夜,次日再置入液氮内(-196℃)低温保存最佳;细胞浓度以2×10~7/ml最好;冻存保护剂以含10％DMSO,90％胎牛血清为宜。
Umbilical cord blood stem / progenitor cells instead of bone marrow transplantation, treatment of radiation-induced damage, and for victims of nuclear accident treatment of cord blood stem cell bank is an important issue, and cord blood stem cell isolation and cryogenic storage is the establishment of cord blood Stem cell bank, the key to stem cell transplantation. The isolation and cryopreservation methods of umbilical cord blood hematopoietic stem cells were studied. Firstly, cord blood mononuclear cells (MNC) were isolated by density gradient centrifugation with different proportions of Ficoll fluid and counted. ) Monoclonal antibody by flow cytometry phenotypic analysis; and then add a cryoprotectant gradient cooling, cryopreservation in liquid nitrogen, and then regularly resuming MNC at regular intervals. The isolation and cryopreservation effects of MNC in cord blood were compared under different conditions using “three gradient cooling methods”, “five different cell concentrations” and “two cell cryoprotectants”. The results showed that the percentage of CD34 + cells in MNC isolated by 1.076 Ficoll solution was higher than that of conventional 1.077 cells. The former was about 3 times higher than the latter, but the total number of cells was less. Nitrogen bottle mouth (liquid phase above the gas phase) overnight, the next day and then placed in liquid nitrogen (-196 ℃) stored at low temperature best; cell concentration to 2 × 10 ~ 7 / ml best; Containing 10% DMSO, 90% fetal bovine serum is appropriate.