Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Butlerova 5A, Moscow 117485, Russia.
Cells. 2023 Apr 29;12(9):1286. doi: 10.3390/cells12091286.
Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications.
磁细胞分选技术因其速度快、操作简单且能够处理大量细胞而脱颖而出。然而，它也存在一些缺点，特别是低分辨力，导致所有或无的选择结果仅限于将细胞群体批量分离为阳性和阴性分数，所选细胞的纯度也不高，而且无法选择表面标记物高表达的细胞亚群。在本研究中，我们开发了一种简单的解决方案来解决这个问题，并通过用磁性分选转导细胞群体的多次实验证实了这种方法的有效性。用共表达荧光报告蛋白 EGFP（增强型绿色荧光蛋白）或 DsRed-Express2 和 LNGFR（低亲和力神经生长因子受体）的双顺反子逆转录病毒载体构建体转导小鼠 NIH 3T3 细胞。评估了抗 LNGFR Micro Bead（MB）剂量范围为 0.5 至 80 μL 对转导细胞的磁性分选的影响。MB 的低剂量有利于从群体中耗尽弱阳性细胞，从而导致所选细胞分数中 EGFP 或 DsRed-Express2 报告基因的表达水平更高。低 MB 剂量还导致所选群体的纯度增加，即使对于初始阳性细胞百分比较低的样本也是如此。同时，高 MB 剂量导致选择后产量增加，并且更忠实于原始表达谱。我们进一步证明，对于表达水平分布相当狭窄的群体，使用基于低剂量和高剂量 MB 顺序使用的两阶段分选方案，有可能将其分离为高表达和低表达亚群。对于表达分布较宽的群体，使用低剂量或高剂量 MB 的单阶段分选足以分别在柱保留和流穿部分清楚地区分低表达和高表达亚群。这项研究极大地扩展了磁细胞分选的潜力，并可能在许多生物医学应用中开辟新的可能性。
