Yamaguchi M, Sawada K, Sato N, Koizumi K, Sekiguchi S, Koike T
Department of Internal Medicine II, Hokkaido University School of Medicine, Japan.
Bone Marrow Transplant. 1997 Feb;19(4):373-9. doi: 10.1038/sj.bmt.1700659.
To achieve a rapid and an efficient purification of CD34+ cells, we devised a nylon-fiber syringe (NF-S) and we manipulated it to deplete adherent cells from normal human blood mononuclear cells (MN cells). The cells processed by NF-S were further purified as the CD34+ fraction, using CD34 monoclonal antibody, immunomagnetic microspheres and chymopapain treatment to detach the microspheres. When steady-state human peripheral blood MN cells were processed by NF-S at 24 degrees C for 5 min, the frequency of monocytes (CD14+ cells) significantly decreased from 22.0 +/- 5.2% to 2.5 +/- 0.4%, with a 73% recovery of CD34+ cells. The subsequent immunomagnetic positive selection achieved preparations of 91 +/- 8% pure CD34+ cells with a 86 +/- 23% yield. The overall yield of CD34+ cells was 44 +/- 11%, and the time required for all procedures was 5 h. There was a tight and an inverse correlation (P < 0.0001) between the frequency of CD14+ cells in the initial cell population and the purity of CD34+ cells in the final preparation. A recommended frequency of CD14+ cells for achieving preparations of over 90% pure CD34+ cells was less than 4.4%. When combining NF-S and immunomagnetic microspheres, efficient bench-top separation of CD34+ cells in steady-state peripheral blood can be done in any laboratory, and fluorescence-activated cell-sorting is not required.
为了快速高效地纯化CD34+细胞，我们设计了一种尼龙纤维注射器（NF-S），并对其进行操作以从正常人血单核细胞（MN细胞）中去除贴壁细胞。使用CD34单克隆抗体、免疫磁珠和木瓜凝乳蛋白酶处理以分离磁珠，将经NF-S处理的细胞进一步纯化为CD34+组分。当在24℃下用NF-S处理稳态人外周血MN细胞5分钟时，单核细胞（CD14+细胞）的频率从22.0±5.2%显著降至2.5±0.4%，CD34+细胞回收率为73%。随后的免疫磁珠阳性分选获得了纯度为91±8%的CD34+细胞制剂，产率为86±23%。CD34+细胞的总产率为44±11%，所有步骤所需时间为5小时。初始细胞群体中CD14+细胞的频率与最终制剂中CD34+细胞的纯度之间存在紧密的负相关（P<0.0001）。为了获得纯度超过90%的CD34+细胞制剂，推荐的CD14+细胞频率低于4.4%。当将NF-S和免疫磁珠结合使用时，任何实验室都可以在台式上高效分离稳态外周血中的CD34+细胞，且无需荧光激活细胞分选。
