Journal: Advanced Science

Article Title: Nanoneedle‐Based Electroporation for Efficient Manufacturing of Human Primary Chimeric Antigen Receptor Regulatory T‐Cells

doi: 10.1002/advs.202416066

Figure Lengend Snippet: Nanoneedle electroporation of Tregs. a) Schematic illustration of the nN‐EP‐system setup. Not to scale. b) Plot of electric field intensity across a vertical line at the interface starting from the nanoneedle (red trace) or flat electrode (blue trace) side to the cell side. Nanoneedle tip = 8.45 kV cm −1 . Control planar surface = 1.38 kV cm −1 . c) Electric field (kV/cm) intensity plot at the nanoneedle tip for the nN‐EP‐system at 10 V, scale bar = 50 nm. d) Electric field (kV/cm) intensity plot for the nN‐EP‐system at 10 V, scale bar = 5 µm. e) Electric field (kV/cm) intensity plot for a flat silicon electrode at 10 V, scale bar = 5 µm. f) Stepwise assembly of the nN‐EP‐system: Stage 1 shows the bottom nanoneedle holder with three square holding areas for holding the nanoneedle chips; Stage 2 shows the nanoneedle chips positioned within the holding areas; Stage 3 shows the PDMS spacer layered over the nanoneedle chips; and Stage 4 shows the fully assembled nanoneedle electroporation well. g) Quantification of cell viability across electroporation conditions. Data presented as mean ± standard deviation (SD), n = 3, one‐way ANOVA followed by Tukey's multiple comparison test. p ‐values are indicated above the bars. h) Quantification of cell viability as a function of electroporation buffer. Data presented as mean ± SD, n = 3, one‐way ANOVA followed by Tukey's multiple comparison test. p ‐values are indicated above the bars. i) SEM image of the Au‐coated nanoneedles. Red arrows indicating gold deposition. Scale bar: 1 µm. j) Viability comparison for cells electroporated under optimized conditions using flat silicon chips (Flat), silicon nanoneedles (nN‐EP Si) and Au‐coated silicon nanoneedles (nN‐EP Au) compared with untreated control (Untreated). Data presented as mean ± SD, n = 3, one‐way ANOVA. k) Quantification of Cy5 + Tregs population indicative of CAR construct delivery for each electroporation condition. Data presented as mean ± SD, n = 3, one‐way ANOVA followed by Tukey's multiple comparison test. p‐ values are indicated above the bars. l) Representative flow cytometry histogram showing Cy5 fluorescence intensity distribution for each electroporation condition compared to untreated control. m) Representative confocal microscopy image showing Cy5 + Treg on nN loaded with Cy5‐tagged plasmid (Red). Treg nucleus was stained with DAPI (Blue). Scale bar: 5 µm.

Article Snippet: Nanoneedle‐EP holders and electrodes were designed using Fusion 360 (Autodesk, USA) and printed using a 3D printer (Form 3+, Formlabs, USA) with medical‐grade BioMed Clear Resin (Formlabs, USA).

Techniques: Electroporation, Control, Standard Deviation, Comparison, Construct, Flow Cytometry, Fluorescence, Confocal Microscopy, Plasmid Preparation, Staining