Date on Master's Thesis/Doctoral Dissertation
5-2024
Document Type
Master's Thesis
Degree Name
M. Eng.
Department
Bioengineering
Degree Program
JB Speed School of Engineering
Committee Chair
Kopechek, Jonathan
Committee Member
Yaddanapudi, Kavitha
Committee Member
Chen, Joseph
Author's Keywords
CAR-T; Jurkat; Acoustofluidic; Sonoporation
Abstract
The continuous pursuit of effective methods for gene transfection in primary human T cells is crucial for advancing cell-based therapies. Viral vectors, currently used in cell-based therapies, involve a lengthy and costly process. This thesis explores the efficacy of coupling acoustofluidic and sonoporation techniques as an alternative non-viral gene transfection approach with primary T cells. Sonoporation, in conjunction with microbubbles, has been demonstrated to induce cavitation, resulting in the formation of pores in the cell membrane through which molecules can enter. Using flow cytometry, we compared the loading efficiency of primary T cells with that of Jurkat T cells to provide essential comparative insights. Employing confocal microscopy allows us to visualize the distribution of fluorescent molecules within the cells to validate successful loading. The study is unique in utilizing confocal fluorescence microscopy to confirm intracellular molecule presence after acoustofluidic sonoporation—an aspect not extensively documented previously. Our results indicate that acoustofluidic sonoporation can successfully deliver fluorescent molecules into the primary T cells, presenting a distributed pattern throughout the cell. However, this efficiency was notably lower when compared to Jurkat T cells. The acoustofluidic and sonoporation coupling was not as successful in delivering fluorescence-expressing plasmids. The study provides an initial framework for considering sonoporation as a viable gene delivery method but also outlines limitations and the necessity for subsequent research. This study's conclusions underscore the effectiveness of acoustofluidic treatment in transfecting primary T cells with fluorescent molecules. Notably, this study contributes to the body of knowledge by elucidating the response of human primary T cells to acoustofluidic transfection, potentially informing the development of more effective non-viral transfection protocols.
Recommended Citation
Colebank, Madison C., "Acoustofluidic delivery of biomolecules to cells: a novel approach for preparing CAR-T cells." (2024). Electronic Theses and Dissertations. Paper 4413.
Retrieved from https://ir.library.louisville.edu/etd/4413
