Todd Sulchek, PhD

Georgia Institute of Technology, Woodruff School of Mechanical Engineering

Thesis Committee:

Alexander Alexeev, PhD

Georgia Institute of Technology, Woodruff School of Mechanical Engineering

Edmund K. Waller, MD, PhD

Emory University School of Medicine, Department of Medical Oncology

Mark Prausnitz, PhD

Georgia Institute of Technology, School of Chemical and Biomolecular Engineering

Krishnendu Roy, PhD

Georgia Institute of Technology, Coulter Department of Biomedical Engineering

CONVECTIVE INTRACELLULAR MACROMOLECULE DELIVERY BY CELL VOLUME EXCHANGE FOR CELL ENGINEERING APPLICATIONS

The rapidly growing field of cell manufacturing requires robust methods for intracellular delivery of cell engineering reagents. However, this field still lacks an intracellular delivery platform that is cost-effective, maintains high cell viability, and is broadly applicable for diverse cargoes and cell types. In this project, we discovered a unique biophysical phenomenon of transient cell volume exchange induced by rapid and sequential microfluidic cell compression. This behavior consists of iterations of brief, mechanically induced cell volume loss followed by rapid volume recovery. We found that cell volume exchange can convectively transfer a variety of macromolecules and particles into cells, including 2 MDa polysaccharides, 100 nm nanoparticles, and 2.5 MDa plasmids, without significantly impacting cell viability or other tested phenotypic properties. The ease of use and successful proof-of-concept transfections demonstrate great potential to address major challenges in intracellular delivery. The work first aims to understand the mechanisms of this technology, and then leverage the platform to impactfully address two growing needs in cell engineering: (1) test and evaluate a new immunotherapy manufacturing approach by delivering CD19 chimeric antigen receptor (CAR) mRNA to primary T cells and assessing CAR T cell expression and cytotoxicity against B cell lymphoma; (2) demonstrate induced pluripotent stem cell (iPSC) reprogramming by direct delivery of Yamanaka factor protein or mRNA.