密度梯度离心法分离骨髓单个核细胞方法的优化

目的:对密度梯度离心法分离骨髓单个核细胞的方法进行优化,以提高该方法分离骨髓单个核细胞的收率。方法:骨髓血40份,100 ml/份,分为常规组和优化组,每组20份。常规组骨髓与NS 1∶1混合稀释后,直接通过Ficoll分离液分离单个核细胞;优化组通过先将骨髓血洗涤一次,加入等量NS充分吹打混匀,200目筛网过滤,保持细胞悬液和Ficoll分离液1∶1的体积比进行分离操作。对每份分离得到的骨髓单个核细胞通过白细胞稀释液稀释后计数,计算单个核细胞的回收率。结果:常规组,分离后的白膜层有12份界面不清晰,同时均混有少量的红细胞。优化组,分离后的白膜层有3份界面不清晰,仅有极少的红细胞混入。两种方法收集的骨髓单个核细胞数比较,优化组多于常规组(P﹤0.01)。结论:通过对密度梯度离心法的步骤进行优化,可提高骨髓单个核细胞的分离效果。

Optimization of the isolation of mononuclear cells from bone marrow by density gradient centrifugation method

Objective We optimized the method of separating bone marrow mononuclear cells by density gradient centrifugation in order to improve the mononuclear cells yield.Methods We selected 40 copies bone marrow,each 40 millilitre,and equally separated into the conventional group and optimized group.The bone marrow in conventional group mixed with normal saline 1∶1 mixture of diluted,directly through the Ficoll separation medium,mononuclear cells;optimize the group through the first bone marrow washed once,add equal amount of the full wind and percussion blending NS,200 mesh filter,keep the cell suspension Ficoll separation medium solution and the volume ratio of 1∶1 for the separation operation.For each isolated from bone marrow mononuclear cells diluted by white blood cells count dilution.Results Conventional group,after the separation of the white film there are 12 interface is not clear,but are mixed with a small amount of red blood cells.Optimize the group,after the separation of the white film has 3 interface is not clear,only very few red blood cells mixed.Two methods to collect the bone marrow mononuclear cells compared to optimize the group than the conventional group(p <0.01).Conclusions We can improve bone marrow mononuclear cells yield by optimizing density gradient centrifugation steps.