Localized delivery of coding nucleic acids into adherent cells by in situ electroporation: integrated impedance-based monitoring allows for loss-of-function or gain-of-function screening

A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In Suspension T Cells and 3D Cell Models.

Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4⁺ and CD8⁺ T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.

An integrated nanopore–microfluidic platform for low-voltage electroporation of delivering self-amplifying RNA into dendritic cells

Efficient delivery of large, negatively charged self-amplifying RNA (saRNA) into dendritic cells (DCs) is critical for next-generation cancer vaccines. However, this remains challenging due to the high sensitivity of DCs to chemical carriers and high-voltage electroporation. In this study, an integrated nanopore-electroporation (NEP) microdevice was developed by combining 200 nm track-etched polycarbonate membrane, bidirectional PDMS microfluidic channels, and Pt/ITO electrodes to localize the electric field and induce membrane permeabilization at low voltage (≤30 V). Multiphysics simulations revealed that 200 nm nanopores concentrated the electric field at the cell–membrane interface, generating transmembrane potentials exceeding 3 V. Using DC2.4, the NEP system achieved 75% propidium iodide (PI) uptake at 25 V with 90% viability, confirming controllable nanoscale perforation. Direct delivery of GFP-encoding saRNA achieved approximately 50% transfection efficiency with sustained protein expression for more than 96 h, significantly outperforming mRNA at an equal dose. Long-term viability (>85% at 96 h) and negligible cytotoxicity demonstrated the excellent biocompatibility of the device. This reagent-minimal, modular NEP platform thus provided a high-efficiency, low-toxicity route for saRNA delivery into hard-to-transfect immune cells, offering a versatile engineering framework for DC-based cancer immunotherapy, RNA vaccine development, and broader cell gene-modification applications.

Microfluidic Electroporation: Recent Advances and Applications.

A novel silicon-based intracellular delivery platform enables multiplexed delivery across cell types and cell states 2310229

Immune cell engineering is a critical element of basic immunological research and translational applications. The ability to deliver modifying materials to cells without substantial phenotypic or survival impacts is a limiting factor for many immune cell types. To enable engineering in sensitive cell types or cell states, we have developed a novel intracellular delivery platform that works through mechanoporation. Cells are pushed through pores on a thin silicon membrane which creates temporary openings in the cell membrane that allow the payload of interest to diffuse into the cytoplasm before the membranes repair themselves. This method has been used to introduce CRISPR RNPs, mRNA, circRNA, siRNA and peptides and is compatible across cell types including naive and activated T cells, B cells, NK cells, monocytes, neutrophils, iPSC and HSC. Using our device, we simultaneously delivered a B2M targeting CRISPR RNP, GFP mRNA, and fluorescent dextran to primary unstimulated T cells and observed that 85% of the live population was B2M negative, GFP and dextran positive. The temporary membrane disruption is gentle enough to use with sensitive cells like neutrophils which allowed us to deliver dextran and GFP mRNA to neutrophils in whole blood. We measured 83% GFP positivity in the live, dextran positive population the next day. Similarly, we delivered a GFP-mRNA to PBMCs and observed expression in T cells, B cells, NK cells, and monocytes. This technology can also enable the use of viral vectors in challenging cell types. Using our method, we engineered B cells to express a circRNA ‘transduction enhancer’ (TE) and then infected them with a GFP lentivirus. After 3 days, the cells that received the TE had the highest GFP expression. Our technology unlocks scalable, gentle, and versatile intracellular delivery for sensitive immune cells, expanding the feasibility of complex genetic and molecular engineering for research and clinical cell therapies. n/a Technological Innovations in Immunology (TECH)

A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos

Direct Cytosolic Delivery of Amphiphilic Framework Nucleic Acids for RNA Interference.

The efficient cytosolic delivery of nucleic acid molecular machines remains a major challenge due to the dual barriers of the cell membrane and endosomal sequestration. Here, we report a class of amphiphilic framework nucleic acids (ampFNAs) that enable direct cytosolic delivery involving energy-independent traversal of lipid membranes. Among several designed geometries, a rigid rod-like six-helix bundle functionalized with a single cholesterol moiety exhibits superior cellular binding and internalization. We find that this ampFNA can enter cells through a cholesterol-dependent, lipid raft-mediated pathway, capable of bypassing endosomal entrapment. Compared to the commercial transfection reagent Lipofectamine 3000 (Lipo3000), the ampFNA platform exhibits reduced lysosomal entrapment. When delivering small interfering RNAs (siRNAs), the ampFNA-mediated enhanced green fluorescent protein (EGFP) gene silencing was achieved with efficiency comparable to Lipo3000. By targeting the proto-oncogene Bcl-2, the ampFNA induced an apoptotic rate of 31.3% in the tumor cell population. Our work establishes ampFNAs as a programmable, efficient, and biocompatible platform for the development of next‑generation smart nucleic acid delivery machines for precision medicine.