Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany ; Department of Pediatric Cardiology, Heart Center GmbH, Faculty of Medicine, University of Leipzig, Leipzig, Germany.
PLoS One. 2013 Sep 6;8(9):e74745. doi: 10.1371/journal.pone.0074745. eCollection 2013.
Isolation of different cell types from one sample by fluorescence activated cell sorting is standard but expensive and time consuming. Magnetic separation is more cost effective and faster by but requires substantial effort. An innovative pluriBead-cascade cell isolation system (pluriSelect GmbH, Leipzig, Germany) simultaneously separates two or more different cell types. It is based on antibody-mediated binding of cells to beads of different size and their isolation with sieves of different mesh-size. For the first time, we validated the pluriSelect system for simultaneous separation of CD4+- and CD8+-cells from human EDTA-blood samples. Results were compared with those obtained by magnetic activated cell sorting (MACS; two steps -first isolation of CD4+, then restaining of the residual cell suspension with anti-human CD8+ MACS antibody followed by the second isolation). pluriSelect separation was done in whole blood, MACS separation on density gradient isolated mononuclear cells. Isolated and residual cells were immunophenotyped by 7-color 9-marker panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLA-DR) using flow cytometry. Cell count, purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (MACS (median[range]: 92.4% [91.5-94.9] vs. pluriSelect 95% [94.9-96.8])) of CD4+ cells, however CD8+ isolation showed lower purity by MACS (74.8% [67.6-77.9], pluriSelect 89.9% [89.0-95.7]). Yield was not significantly different for CD4 (MACS 58.5% [54.1-67.5], pluriSelect 67.9% [56.8-69.8]) and for CD8 (MACS 57.2% [41.3-72.0], pluriSelect 67.2% [60.0-78.5]). Viability was slightly higher with MACS for CD4+ (98.4% [97.8-99.0], pluriSelect 94.1% [92.1-95.2]) and for CD8+-cells (98.8% [98.3-99.1], pluriSelect 86.7% [84.2-89.9]). pluriSelect separation was substantially faster than MACS (1h vs. 2.5h) and no pre-enrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two and more cell subpopulation directly from whole blood and provides a simple alternative to magnetic separation.
通过荧光激活细胞分选从一个样本中分离不同的细胞类型是标准的，但昂贵且耗时。磁分离更具成本效益且速度更快，但需要大量的努力。一种创新的多珠级联细胞分离系统（pluriSelect GmbH，莱比锡，德国）可以同时分离两种或更多不同的细胞类型。它基于抗体介导的细胞与不同大小的珠子结合，并使用不同网眼尺寸的筛子进行分离。我们首次验证了 pluriSelect 系统用于同时分离人 EDTA 血液样本中的 CD4+和 CD8+细胞。结果与通过磁性激活细胞分选（MACS；两步法 - 首先分离 CD4+，然后用抗人 CD8+MACS 抗体重新染色剩余细胞悬浮液，然后进行第二次分离）获得的结果进行了比较。pluriSelect 分离是在全血中进行的，MACS 分离是在密度梯度分离的单核细胞上进行的。使用流式细胞术通过 7 色 9 标志物面板（CD3；CD16/56；CD4；CD8；CD14；CD19；CD45；HLA-DR）对分离和残留细胞进行免疫表型分析。细胞计数、纯度、产量和活力（7-AAD 排除）。两种系统在 CD4+细胞的纯度方面没有显著差异（MACS（中位数[范围]：92.4%[91.5-94.9] vs. pluriSelect 95%[94.9-96.8]），然而，MACS 对 CD8+的分离显示出较低的纯度（74.8%[67.6-77.9]，pluriSelect 89.9%[89.0-95.7]）。对于 CD4（MACS 58.5%[54.1-67.5]，pluriSelect 67.9%[56.8-69.8]）和 CD8（MACS 57.2%[41.3-72.0]，pluriSelect 67.2%[60.0-78.5]），产量没有显著差异。对于 CD4+（MACS 98.4%[97.8-99.0]，pluriSelect 94.1%[92.1-95.2]）和 CD8+-细胞（MACS 98.8%[98.3-99.1]，pluriSelect 86.7%[84.2-89.9]），MACS 的活力略高。pluriSelect 分离速度明显快于 MACS（1 小时对 2.5 小时），并且不需要预富集步骤。总之，pluriSelect 是一种快速、简单、温和的系统，可直接从全血中高效同时分离两种或更多细胞亚群，并为磁分离提供了简单的替代方案。
