Intracellular Delivery of Exogenous Macromolecules into Human Mesenchymal Stem Cells by Double Deformation of the Plasma Membrane

Physical techniques for intracellular delivery of exogeneous materials offer an attractive strategy to enhance the therapeutic efficiency of stem cells. However, these methods are currently limited by poor delivery efficiency as well as cytotoxic effects. Here, a high throughput microfluidic device is designed for efficient (≈85%) cytosolic delivery of exogenous macromolecules with minimal cell death (less than 10%). The designed microfluidic device enables the generation of transient pores as the cells pass through the micron‐sized constrictions (6–10 µm) leading to the passive diffusion of extracellular cargos into the cell cytosol. Specifically, the microfluidic system is designed to induce a double deformation on the cell membrane at the squeezing zones to maximize intracellular delivery. Additionally, the flow rate, ionic concentration, and the molecular weight of the cargo are optimized for maximum efficiency. The optimized device enables cytosolic diffusion of small (3 kDa) and large molecules (70 kDa) without inducing any apoptotic effect. Overall, this double cell deformation platform offers new opportunities to rapidly and efficiently deliver extracellular cargo into stem cells without affecting their viability and functionality. The designed microfluidic device can promote efficient deformation of the cell membrane to promote the internalization of extracellular macromolecules within the cell. The structure of the chip can be varied to promote a single and a double deformation of the cell membrane for each squeezing path. The double deformation design allows a higher internalization of the extracellular cargo.