On-chip multiplexed single-cell patterning and controllable intracellular delivery

Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy. 3D Electroporation: Uniform and safe electricity-based drug delivery Applying an electric field to individually trapped cells allows for the efficient and safe delivery of drugs or genes, and the characterization and manipulation of cellular activity. Electroporation is a technique that increases the permeability of a cell’s membrane, to allow the infiltration of drugs or genes. In practice, traditional electroporation has issues with efficiency and cellular damage. The scientists of Lingqian Chang (Beihang University) and Mo Li (Peking University Third Hospital) led a team to develop an on-chip “3D” system that uses a vacuum to trap cells in individual micropores. By applying an electric field, the team were able to deliver cargo of different sizes, from drugs to genes, into the cells with greater efficiency and safety than benchmark methods. The dose of chemotherapy drugs was controlled through altering the voltage applied. The team were able to inhibit tumor activity by delivering CRISPR-Cas9 gene editing plasmid with