Nanochannel Electro‐Injection as a Versatile Platform for Efficient RNA/DNA Programming on Dendritic Cells

Liu, Jing; José, Juán; Deng, Caiguanxi; Huang, Xinshuo; Huang, Shuang; Liu, Zhengjie; Yang, Jianguo; Mo, Jingshan; Chen, Hui‐Jiuan; Wang, Ji; Xie, Xi

doi:10.1002/smll.202303088

Cited by 5 publications

(11 citation statements)

References 57 publications

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“…Given the significance of the transmembrane potential in facilitating cell membrane perforation, we solely manipulate voltage to optimize transfection efficiency and cell viability while maintaining the constancy of other parameters of the electric pulses, including pulse frequency, pulse width, and duration of electric stimulation. Consistent with our previous work, the electric pulses are maintained with a frequency of 20 Hz, a pulse width of 200 μs, and a total duration of electric stimulation at 30 s Based on these established parameters, the voltage of the electric pulse is gradually increased from 10 to 35 V to facilitate the transfection process within HeLa cells. After the electric stimulation, the cells are allowed to undergo a stabilization period of 15 min.…”

Section: Resultssupporting

confidence: 56%

“…Consequently, it becomes challenging to directly examine the morphology of the resulting nanopores by electron microscopy, since it normally needs chemical fixation of the cells at the moment immediately following the perforation event. Previous research has demonstrated that cell membrane perforations induced through this method are typically fully repaired within a maximum of 5 min, thereby producing less desirable impact on cellular viability . To achieve the delivery of nucleic acid molecules, the Pt electrode in the cell culture is connected to the anode of the power supply (left devices in Figure g), while the ITO glass electrode is connected to the cathode.…”

Section: Resultsmentioning

confidence: 99%

“…The manufacturing protocol of NPE devices has been detail demonstrated in our previous work. 36 In brief, the NPE device is composed of a cellculture chamber made of PDMS, a middle layer of NPM, a thin PDMS layer with a microfluidic channel, and ITO glass, as shown in Figure S1. The manufacturing process involves mixing PDMS monomer and curing agent (Dow Corning) at a volume ratio of 10 (monomer):1 (curing agent), followed by pouring the mixture into a mold and curing at 80 °C for at least 30 min.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The other region of the cell membrane remains intact, because the transmembrane potential does not reach the threshold, resulting in localized electroporation of the cell Previous research has demonstrated that cell membrane perforations induced through this method are typically fully repaired within a maximum of 5 min, thereby producing less desirable impact on cellular viability. 36 To achieve the delivery of nucleic acid molecules, the Pt electrode in the cell culture is connected to the anode of the power supply (left devices in Figure 1g), while the ITO glass electrode is connected to the cathode. This particular electrode configuration accounts for the negatively charged DNA molecules, enabling their efficient migration along the nanopore channel toward the cells aided by electrophoresis.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Our previous work mainly focused on enhancing transfection efficiency by manipulating the parameters of the nanopores and electric fields, paying relatively less attention to the growth characteristics of the cells themselves. 36 As a result, there is a lack of comprehensive and systematic investigations regarding the applicability of nanopore electroporation technology across various cell lines. In the realm of clinical applications, where the manipulation and genetic modification of intricate cell types such as stem cells, immune cells, and neural cells are frequently encountered, the enhancement of transfection efficiency necessitates the meticulous optimization of transfection conditions aligned with the specific growth attributes of these cells.…”

Section: ■ Introductionmentioning

confidence: 99%

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Nanopore Electroporation Device for DNA Transfection into Various Spreading and Nonadherent Cell Types

Liu,

Jiang,

et al. 2023

ACS Appl. Mater. Interfaces

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Cell transfection plays a crucial role in the study of gene function and regulation of gene expression. The existing gene transfection methods, such as chemical carriers, viruses, electroporation, and microinjection, suffer from limitations, including cell type dependence, reliance on cellular endocytosis, low efficiency, safety concerns, and technical complexity. Nanopore-coupled electroporation offers a promising approach to localizing electric fields for efficient cell membrane perforation and nucleic acid transfection. However, the applicability of nanopore electroporation technology across different cell types lacks a systematic investigation. In this study, we explore the potential of nanopore electroporation for transfecting DNA plasmids into various cell types. Our nanopore electroporation device employs track-etched membranes as the core component. We find that nanopore electroporation efficiently transfects adherent cells, including well-spreading epithelial-like HeLa cells, cardiomyocyte-like HL-1 cells, and dendritic-cell-like DC2.4 cells. However, it shows a limited transfection efficiency in weakly spreading macrophages (RAW264.7) and suspension cells (Jurkat). To gain insights into these observations, we develop a COMSOL model, revealing that nanopore electroporation better localizes the electric field on adherent and well-spreading cells, promoting favorable membrane poration conditions. Our findings provide valuable references for advancing nanopore electroporation as a high-throughput, safe, and efficient gene transfection platform.

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Section: Resultssupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Nanopore Electroporation Device for DNA Transfection into Various Spreading and Nonadherent Cell Types

Liu,

Jiang,

et al. 2023

ACS Appl. Mater. Interfaces

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Automating CAR‐T Transfection with Micro and Nano‐Technologies

Hu,

Kumar,

Luo

et al. 2023

Small Methods

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Cancer poses a significant health challenge, with traditional treatments like surgery, radiotherapy, and chemotherapy often lacking in cell specificity and long‐term curative potential. Chimeric antigen receptor T cell (CAR‐T) therapy,utilizing genetically engineered T cells to target cancer cells, is a promising alternative. However, its high cost limits widespread application. CAR‐T manufacturing process encompasses three stages: cell isolation and activation, transfection, and expansion.While the first and last stages have straightforward, commercially available automation technologies, the transfection stage lags behind. Current automated transfection relies on viral vectors or bulk electroporation, which have drawbacks such as limited cargo capacity and significant cell disturbance. Conversely, micro and nano‐tool methods offer higher throughput and cargo flexibility, yet their automation remains underexplored.In this perspective, the progress in micro and nano‐engineering tools for CAR‐T transfection followed by a discussion to automate them is described. It is anticipated that this work can inspire the community working on micro and nano transfection techniques to examine how their protocols can be automated to align with the growing interest in automating CAR‐T manufacturing.

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