UC Irvine

In recent years, cellular and gene therapies have been transforming medicine. With the 2018 Nobel Prize awarded to pioneers in the field of cancer immunotherapy, more and more advances in cell engineering are being developed to produce genetic-modified and reprogrammed cells for cellular and gene therapies. One promising category is ex-vivo cell/gene therapy in which the target cells are isolated from patients, the therapy is administrated to the cells outside of the body in vitro, and the cells are then transferred back into the body. However, challenges remain in terms of (i) isolating the target cells to be engineered, (ii) developments of efficient, safe, and controllable methods for intracellular delivery of gene-editing cargos, and (iii) development of efficient quality control (QC) approaches based on single-cell analysis of engineered cells. This dissertation is set out to develop microfluidic technologies to address the challenges in cell engineering and analysis.First, a high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electrical Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with uniform poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. With this platform, we demonstrated large-plasmid (>9kbp) transfection for CRISPR-Cas9 at 1 million cells/min per single chip. Second, toward development of more efficient 1–1 droplet encapsulation methods for single-cell analysis, the mechanism of particle trapping and release at the flow-focusing microfluidic droplet generation junction, utilizing the hydrodynamic micro-vortices generated in the dispersed phase, is described. This technique is based solely on our unique flow-focusing geometry and the flow control of the two immiscible phases and, thus, does not require any on-chip active components. The effectiveness of this technique to be used for particle trapping and the subsequent size selective release into the droplets depends on the fundamental understanding of the nature of the vortex streamlines. Therefore, theoretical, computational, and experimental fluid dynamics were utilized to study in detail these micro-vortices and parameters affecting their formation, trajectory, and magnitude. Third, an integrated microfluidic platform is presented that provides 3-part differential sorting of WBCs from whole blood. The proposed system accomplishes 3-part differential sorting of WBCs by: (1) On-chip lysis of RBCs from the blood sample, and (2) Downstream isolation of subpopulation of WBCs using dielectrophoresis (DEP) technology. The developed platform is capable of efficient isolation of viable monocytes, granulocytes, and lymphocytes from undiluted whole blood sample with volumes as low as 50 ul.