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利用纳米吸管/纳米孔辅助电穿孔实现高效的克隆β细胞转染。

Achieving efficient clonal beta cells transfection using nanostraw/nanopore-assisted electroporation.

作者信息

Ekstrand Frida, Mapar Mokhtar, Ruhrmann Sabrina, Bacos Karl, Ling Charlotte, Prinz Christelle N

机构信息

Division of Solid State Physics, NanoLund, Lund University 221 00 Lund Sweden

Epigenetics and Diabetes Unit, Lund University Diabetes Centre, Department of Clinical Sciences, Scania University Hospital 214 28 Malmö Sweden.

出版信息

RSC Adv. 2024 Jul 15;14(31):22244-22252. doi: 10.1039/d4ra02791d. eCollection 2024 Jul 12.

Abstract

The prospect of being able to efficiently inject large plasmids in insulin-producing beta cells is very attractive for diabetes research. However, conventional transfection methods suffer from high cytotoxicity or low transfection efficiency, which negatively affect their outcome. In contrast, nanostraw electroporation is a gentle method that can provide a high transfection efficiency while maintaining high cell viability. While nanostraw electroporation has gone through some method optimization in the past, such as tuning the pulse frequency, amplitude, and duration, the effect of other parameters has not been thoroughly investigated. Here, we demonstrate efficient transfection of clonal beta cells and investigate the effect of voltage at a fixed inter-electrode distance, cell density, and cargo solution conductivity on transfection efficiency. We used GFP-encoding DNA plasmids stained with an intercalating dye to enable immediate analysis and assessment of the electrophoretic transport of cargo. Moreover, we ran simulations to assess how cargo buffer conductivity impacts the transfection efficiency by affecting the voltage drop on the nanostraws and cell membrane during electroporation. Both experiments and simulations show that MilliQ water as the cargo buffer yields the best transfection efficiency. We also show that the cell density should be adjusted to maximize the number of cells interfacing the nanostraws and avoid cell stacking. Finally, we compared the transfection efficiency when using nanostraws and nanopores. Whereas the amount of GFP plasmids injected using nanostraws is larger than for nanopores, the outcome in terms of GFP fluorescence 48 h after transfection was worse than for nanopores. Moreover, when using nanostraws, fewer cells were found on the substrate 48 h after transfection compared to when using nanopores. This suggests that injecting substantial amounts of plasmids in cells can affect their proliferation and/or viability, and that nanopore electroporation, as a simpler method, is an interesting alternative to nanostraws in achieving efficient and gentle clonal beta cell transfection.

摘要

能够在产生胰岛素的β细胞中高效注射大质粒的前景对于糖尿病研究非常有吸引力。然而,传统的转染方法存在高细胞毒性或低转染效率的问题,这对其结果产生负面影响。相比之下,纳米吸管电穿孔是一种温和的方法,能够在保持高细胞活力的同时提供高转染效率。虽然纳米吸管电穿孔在过去已经经历了一些方法优化,如调整脉冲频率、幅度和持续时间,但其他参数的影响尚未得到充分研究。在这里,我们展示了克隆β细胞的高效转染,并研究了在固定电极间距、细胞密度和载液电导率下电压对转染效率的影响。我们使用用嵌入染料染色的编码绿色荧光蛋白(GFP)的DNA质粒,以便能够立即分析和评估货物的电泳运输。此外,我们进行了模拟,以评估货物缓冲液电导率如何通过影响电穿孔过程中纳米吸管和细胞膜上的电压降来影响转染效率。实验和模拟均表明,以超纯水作为货物缓冲液可产生最佳转染效率。我们还表明,应调整细胞密度,以最大限度地增加与纳米吸管接触的细胞数量并避免细胞堆积。最后,我们比较了使用纳米吸管和纳米孔时的转染效率。虽然使用纳米吸管注射的GFP质粒数量比纳米孔多,但转染后48小时的GFP荧光结果比纳米孔差。此外,与使用纳米孔相比,使用纳米吸管时,转染后48小时在底物上发现的细胞较少。这表明在细胞中注射大量质粒会影响其增殖和/或活力,并且作为一种更简单的方法,纳米孔电穿孔在实现高效且温和的克隆β细胞转染方面是纳米吸管的一个有趣替代方案。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/710b/11247384/bd1ebbf0cc21/d4ra02791d-f1.jpg

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