Microfluidic Cell Stretching for Highly Effective Gene Delivery into Hard-to-Transfect Primary Cells

Abstract

Cell therapy and cellular engineering begin with internalizing synthetic biomolecules and functional nanomaterials into primary cells. Conventionally, electroporation, lipofection, or viral transduction has been used; however, these are limited by their cytotoxicity, low scalability, cost, and/or preparation complexity, especially in primary cells. Thus, a universal intracellular delivery method that outperforms the existing methods must be established. Here, we present a versatile intracellular delivery platform that leverages intrinsic inertial flow developed in a T-junction microchannel with a cavity. The elongational recirculating flows exerted in the channel substantially stretch the cells, creating discontinuities on cell membranes, thereby enabling highly effective internalization of nanomaterials, such as plasmid DNA (7.9 kbp), mRNA, siRNA, quantum dots, and large nanoparticles (300 nm), into different cell types, including hard-to-transfect primary stem and immune cells. We identified that the internalization mechanism of external cargos during the cell elongation–restoration process is achieved by both passive diffusion and convection-based rapid solution exchange across the cell membrane. Using fluidic cell mechanoporation, we demonstrated a transfection yield superior to that of other state-of-the-art microfluidic platforms as well as current benchtop techniques, including lipofectamine and electroporation. In summary, the intracellular delivery platform developed in the present study enables a high delivery efficiency (up to 98%), easy operation (single-step), low material cost (<$1), high scalability (1 × 106 cells/min), minimal cell perturbation (up to 90%), and cell type/cargo insensitive delivery, providing a practical and robust approach anticipated to critically impact cell-based research.

Materials for Improving Immune Cell Transfection

“…Reproduced with permission. [ 79 ] Copyright 2020, American Chemical Society. d) Acoustic pressure waves applied to cells flowing through microfluidic channels permeabilized cell membrane for delivery of biomolecules into nuclei at a rate of 200 000 cells min −1 .…”

Microfluidic and Nanofluidic Intracellular Delivery

“…To develop a platform with near‐zero risk of channel clogging and higher delivery performance, the same group reported a unique T‐junction microchannel with a microcavity structure (Figure 7f). [ 15 ] The cavity structure was introduced to exert recirculating flows developed in the T‐junction at moderate Reynolds numbers, which substantially mitigates channel clogging. Since cell mechanoporation was conducted by sequential physical cell‐wall collision and fluid shearing via recirculating vortices, highly efficient delivery of diverse nanomaterials (e.g., 2000 kDa FITC–dextran, mRNA, siRNA, 7.9 kbp plasmid DNA, and 300 nm nanoparticles) into various cell types, including clinical primary cells, was achieved.…”

Single‐Cell Immunoblotting based on a Photoclick Hydrogel Enables High‐Throughput Screening and Accurate Profiling of Exogenous Gene Expression

“…[6] Notably, foreign genes are not integrated into the target cell's chromosome genome in transient transfection method. [7] The expression products of exogenous genes show considerable instability Fast and accurate profiling of exogenous gene expression in host cells is crucial for studying gene function in cellular and molecular biology, but still faces the challenge of incomplete co-expression of reporter genes and target genes. Here, a single-cell transfection analysis chip (scTAC) is presented, which is based on the in situ microchip immunoblotting method, for rapid and accurate analysis of exogenous gene expression in thousands of individual host cells.…”

“…[ 6 ] Notably, foreign genes are not integrated into the target cell's chromosome genome in transient transfection method. [ 7 ] The expression products of exogenous genes show considerable instability during different cell phases, presenting highly dynamic characteristics in transfection process. [ 8 ] Therefore, incomplete co‐expression phenomenon of the fusion gene limits the utility of this strategy in signal transduction regulation mechanism study.…”