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 ≈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 ... More
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 ≈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 ≈5% 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.
