Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department for Plastic Surgery and Hand Surgery; Klinikum rechts der Isar; Technical University Munich, Munich, Germany.
Int J Med Sci. 2017 Jul 19;14(9):798-803. doi: 10.7150/ijms.19241. eCollection 2017.
Dermal fibroblast is a powerful tool for the study of DNA delivery in development of both cell therapy and tissue engineering products. Using genetic modification, fibroblasts can be diversely adapted and made suitable for clinical gene therapy. In this study, we first compared several non-viral transfection methods including nucleofection in rat and human primary dermal fibroblast. In addition, the original protocol for nucleofection of primary mammalian fibroblasts was modified in order to achieve the highest possible transfection efficiency, as determined by flow cytometry analysis of the green fluorescent protein (GFP) expression. : the results showed that transfection performance of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) yielded the best transfection efficiency with rat dermal fibroblasts and ITS (insulin, transferrin, and sodium selenite solution) was comparable to the standard nucleofection solution for human dermal fibroblasts. Our results suggest a promising application of the modified nucleofection method for GMP compatible therapeutic translational medical research.
皮肤成纤维细胞是研究 DNA 传递的有力工具，它在细胞治疗和组织工程产品的开发中都有应用。通过遗传修饰，成纤维细胞可以被多样化地适应，并适合于临床基因治疗。在这项研究中，我们首先比较了几种非病毒转染方法，包括核转染在大鼠和人原代皮肤成纤维细胞中的应用。此外，为了达到最高的转染效率，我们对原代哺乳动物成纤维细胞的核转染方案进行了修改，通过流式细胞术分析绿色荧光蛋白（GFP）的表达来确定。结果表明，在含有 10%胎牛血清（FCS）的 DMEM 培养基中进行转染，可获得最佳的转染效率，且大鼠皮肤成纤维细胞的转染效果优于人皮肤成纤维细胞的标准核转染液。我们的结果表明，改良的核转染方法有望应用于符合 GMP 要求的治疗性转化医学研究。
