Lipoplex-Mediated Single-Cell Transfection via Droplet Microfluidics

While lipoplex (cationic lipid-nucleic acid complex)-mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only approximate to 5% or even lower. Here, efficient and consistent lipoplex-mediated transfection is demonstrated for hard-to-transfect suspension cells via a single-cell, droplet-microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co-confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch-off junction. The transfection efficiency, examined by the delivery of the pcDNA3-EGFP plasmid, improves from approximate to 5% to approximate to 50% for all three tested suspension cell lines, i.e., K562, THP-1, Jurkat, and with significantly reduced cell-to-cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)-CAS9 (CRISPR-associated nuclease 9) mechanism is also achieved using this platform. Lipoplex-mediated single-cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell-to-cell variation for hard-to-transfect suspension cells.