Convective intracellular macromolecule delivery for cell engineering applications

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Liu, Anna
Sulchek, Todd A.
Roy, Krishnendu
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Efficient intracellular delivery of target macromolecules remains a major obstacle in cell engineering, cell labeling, and other biomedical applications. Current standard methods of intracellular delivery, such as viral transduction and electroporation, do not meet the growing needs in the cell engineering field for cost-effective, scalable, and efficient delivery that maintains cell viability. This thesis work has discovered the cell biophysical phenomenon of convective intracellular macromolecule delivery using mechanically-induced, transient cell volume exchange. Ultrafast microfluidic cell compressions (<1 ms) are used to cause brief, deformation-induced cell volume loss followed by volume recovery through uptake of extracellular fluid. Macromolecules suspended in the surrounding fluid enter the cell on convective fluid currents. Convective delivery is shown to bypass endosomal transport and is capable of achieving high intracellular delivery for a broad range of molecule types and sizes. Cell volume exchange is shown to be dependent on strain rate, magnitude of compression, and cell physical properties. The results of this thesis have informed the design and optimization of a high-throughput microfluidic technology capable of efficiently delivering a wide variety of macromolecule payloads to various cell types while maintaining viability and proliferation. We harness this cell volume exchange behavior for convective intracellular delivery of large macromolecules of interest, including plasmids (>2 MDa) and particles (>30 nm), while maintaining high cell viability (>95%). Successful experiments in CRISPR-Cas9 gene editing and intracellular gene expression analysis demonstrate potential to overcome the most prohibitive challenges in intracellular delivery for cell engineering.
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