Kawano Y, Takaue Y, Law P, Watanabe T, Abe T, Okamoto Y, Makimoto A, Sato J, Nakagawa R, Kajiume T, Hirao A, Watanabe A, Kuroda Y
Department of Pediatrics, The University of Tokushima, Japan.
Bone Marrow Transplant. 1998 Nov;22(10):1011-7. doi: 10.1038/sj.bmt.1701479.
CD34+ cells were purified in bulk from apheresis-collected cells of children with cancer using monoclonal antibody (MoAb) and magnetic beads (Baxter ISOLEX system). To improve the purity of the final product for possibly better tumor cell purging and to make the manufacturer's original procedure more cost-effective, we incubated the cells for 30 min with l-phenylalanine methylester hydrochloride (PME) to reduce the cell number by removing contaminating granulocytes and monocytes in the initial step before incubation with MoAb. Our modification prevented nonspecific interactions between MoAb and magnetic beads, and thereby saved expensive materials for purification. A total of 40 purifications were performed with samples containing a mean of 3.1 x 10(9) blood cells mobilized from 15 children by chemotherapy plus granulocyte colony-stimulating factor (G-CSF). The entire purification procedure, from the end of apheresis to storage, was completed within 5h. After incubation with PME and double-layered (40/60%) Percoll separation, the number of CD34+ cells was reduced to 48+/-29%, which suggests the possibility that half of the CD34+ cells in the inoculum were nonclonogenic in the hematopoietic progenitor assay. PME/Percoll-treated cells were then subjected to a final isolation procedure with MoAb according to the manufacturer's suggestions, and 52+/-42% and 32+/-22%, respectively, of the CFU-GM and CD34+ cells present in the initial bag inoculums were recovered. The recovery rates were, respectively, 54% and 67%, when the calculation was limited to the isolation procedure with MNoAb. The purity of isolated CD34+ cells and the plating efficiency in methylcellulose culture were, respectively, 77+/-24% and 33+/-13%. Fourteen children were subsequently autografted with purified CD34+ cells after marrow ablative chemotherapy. The median number of days to achieve an ANC of 0.5 x 10(9)/l was 12 and that to achieve a platelet count of 50 x 10(9)/l was 22.5, which were comparable to those in our historical group of 55 patients who underwent transplant with unmanipulated blood cells (13 and 16 days). These results suggest that our modified purification procedure with PME is useful for the initial reduction of cell numbers to save costly materials, and that cells isolated by this procedure can be directly used in clinical transplantation procedures.
使用单克隆抗体（MoAb）和磁珠（百特ISOLEX系统）从癌症患儿经单采收集的细胞中批量纯化CD34+细胞。为提高最终产品的纯度以可能更好地清除肿瘤细胞，并使制造商的原始程序更具成本效益，我们在与MoAb孵育前的初始步骤中，用盐酸L-苯丙氨酸甲酯（PME）将细胞孵育30分钟，以通过去除污染的粒细胞和单核细胞来减少细胞数量。我们的改进防止了MoAb与磁珠之间的非特异性相互作用，从而节省了昂贵的纯化材料。对来自15名儿童经化疗加粒细胞集落刺激因子（G-CSF）动员的平均含有3.1×10⁹个血细胞的样本进行了总共40次纯化。从单采结束到储存的整个纯化过程在5小时内完成。在用PME孵育和双层（40/60%）Percoll分离后，CD34+细胞数量减少到48±29%，这表明接种物中一半的CD34+细胞在造血祖细胞测定中可能是非克隆性的。然后根据制造商的建议，用MoAb对经PME/Percoll处理的细胞进行最终分离程序，初始袋接种物中分别回收了52±42%和32±22%的CFU-GM和CD34+细胞。当计算仅限于用MoAb的分离程序时，回收率分别为54%和67%。分离的CD34+细胞的纯度和在甲基纤维素培养中的接种效率分别为77±24%和33±13%。随后，14名儿童在骨髓清除化疗后接受了纯化的CD34+细胞自体移植。达到中性粒细胞绝对计数0.5×10⁹/L的中位天数为12天，达到血小板计数50×10⁹/L的中位天数为22.5天，这与我们历史上55名接受未处理血细胞移植的患者组（13天和16天）相当。这些结果表明，我们用PME改进的纯化程序对于初始减少细胞数量以节省昂贵材料是有用的，并且通过该程序分离的细胞可直接用于临床移植程序。
