Muslimov Albert R, Timin Alexander S, Petrova Aleksandra V, Epifanovskaya Olga S, Shakirova Alena I, Lepik Kirill V, Gorshkov Andrey, Il'inskaja Eugenia V, Vasin Andrey V, Afanasyev Boris V, Fehse Boris, Sukhorukov Gleb B
First Pavlov State Medical University of St. Petersburg, Lev Tolstoy str., 6/8, 197022 St. Petersburg, Russian Federation.
Research Institute of Influenza, Popova str., 15/17, 197376 Saint-Petersburg, Russian Federation.
ACS Biomater Sci Eng. 2017 Oct 9;3(10):2314-2324. doi: 10.1021/acsbiomaterials.7b00482. Epub 2017 Sep 22.
Stem cell engineering-the manipulation and functionalization of stem cells involving genetic modification-can significantly expand their applicability for cell therapy in humans. Toward this aim, reliable, standardized, and cost-effective methods for cell manipulation are required. Here we explore the potential of magnetic multilayer capsules to serve as a universal platform for nonviral gene transfer, stem cell magnetization, and magnetic cell separation to improve gene transfer efficiency. In particular, the following experiments were performed: (i) a study of the process of internalization of magnetic capsules into stem cells, including capsule co-localization with established markers of endo-lysosomal pathway; (ii) characterization and quantification of capsule uptake with confocal microscopy, electron microscopy, and flow cytometry; (iii) intracellular delivery of messenger RNA and separation of gene-modified cells by magnetic cell sorting (MACS); and (iv) analysis of the influence of capsules on cell proliferation potential. Importantly, based on the internalization of magnetic capsules, transfected cells became susceptible to external magnetic fields, which made it easy to enrich gene-modified cells using MACS (purity ∼95%), and also to influence their migration behavior. In summary, our results underline the high potential of magnetic capsules in stem cell functionalization, namely (i) to increase gene-transfer efficiency and (ii) to facilitate enrichment and targeting of transfected cells. Finally, we did not observe a negative impact of the capsules used on the proliferative capacity of stem cells, proving their high biocompatibility.
干细胞工程——涉及基因修饰的干细胞操作与功能化——能够显著拓展其在人类细胞治疗中的应用范围。为实现这一目标，需要可靠、标准化且具有成本效益的细胞操作方法。在此，我们探索磁性多层胶囊作为非病毒基因传递、干细胞磁化及磁性细胞分离通用平台以提高基因传递效率的潜力。具体而言，进行了以下实验：（i）研究磁性胶囊内化入干细胞的过程，包括胶囊与内吞溶酶体途径既定标志物的共定位；（ii）利用共聚焦显微镜、电子显微镜和流式细胞术对胶囊摄取进行表征和定量；（iii）通过磁性细胞分选（MACS）进行信使核糖核酸的细胞内递送及基因修饰细胞的分离；以及（iv）分析胶囊对细胞增殖潜能的影响。重要的是，基于磁性胶囊的内化，转染细胞变得易受外部磁场影响，这使得利用MACS富集基因修饰细胞变得容易（纯度约95%），并且还能影响其迁移行为。总之，我们的结果突显了磁性胶囊在干细胞功能化方面的巨大潜力，即（i）提高基因传递效率以及（ii）促进转染细胞的富集和靶向。最后，我们未观察到所用胶囊对干细胞增殖能力有负面影响，证明了它们具有高度生物相容性。
