Towards the development of a continuous-flow, smart micro-electroporation technology to advance cell therapy

FDA approved patient-derived cellular therapies are a groundbreaking biomedical/clinical accomplishment in recent years. This therapy involves the intricate process of removing cells from the patient, genetically modifying them ex vivo, and then returning the cells to the patient to combat disease. Though this field is very promising for treatment of otherwise untreatable cancers and other genetic/auto-immune disorders, current manufacturing costs may make this life-saving therapy unaffordable to the general population. Of the manufacturing steps, the use of viral vectors for gene delivery remains the ‘rate-limiting’ step from an economic point of view. Electroporation is an alternative to viral-mediated gene delivery. Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. This dissertation focuses on advancing a novel, micro-electroporation technology capable of electrically monitoring the degree of cell membrane permeabilization throughout the process. Technological advancements are made from a biochemical standpoint, through optimization of the electroporation buffer that cells are suspended in during the electroporation process, as well as microfluidic/hardware/software design. Highly desirable biomedical and clinical applications, such as DNA plasmid delivery and gene editing with CRISPR-Cas9, are demonstrated using this micro-electroporation technology. Furthermore, ideas to enhance the overall throughput of the technology are introduced, such single-cell level feedback control, population-based feedback control, and microfluidic device parallelization. Ultimately with further technological advancement, this continuous-flow, micro-electroporation system may shift the existing cell therapy manufacturing paradigm, with hopes of eliminating the need for viral-mediated gene delivery.Ph.D.Includes bibliographical reference