Date on Master's Thesis/Doctoral Dissertation

5-2022

Document Type

Master's Thesis

Degree Name

M. Eng.

Department

Bioengineering

Degree Program

JB Speed School of Engineering

Committee Chair

Kopechek, Jonathan

Committee Member

Soucy, Patricia

Committee Member

Yaddanapudi, Kavitha

Author's Keywords

CAR-T; CRISPR/Cas9; cell therapy; non-viral transfection; sonoporation

Abstract

Cell-based immunotherapies are a new generation of “living-drug” treatments for cancer and other diseases. Chimeric antigen receptor (CAR) T-cell cancer therapy has shown promising results in lymphoma and B-cell malignancies. Currently, there are six FDA-approved CAR-T drugs on the market, and all of them use viral transfection for reprogramming. While viral transfection is effective, there are safety concerns due to inconsistent transfection that limit the use of CAR-T therapy. Current non-viral transfection techniques generally have lower transfection efficiency than viral transfection. Additionally, these techniques can be toxic, time-consuming, non-transportable, and expensive. To address these limitations, a novel 3D printed acoustofluidic system is being explored for inducing intracellular molecular delivery of therapeutic compounds for genetic editing. This system utilizes sonoporation, which creates reversible pores in the cell membrane when ultrasound waves rupture microbubbles. A solution of cells, microbubbles, and molecules of interest pass through fluidic channels and microbubbles rupture once exposed to ultrasound waves. A shockwave is created, which propels nearby material into the cell within seconds. In this thesis, several studies were conducted to assess the acoustofluidic mediated intracellular molecular delivery of compounds such as 150kDa FITC-Dextran, fluorescently labeled tracrRNA, and green fluorescent protein (GFP) in multiple cell lines. These studies indicate that cationic microbubbles alongside acoustofluidic treatment significantly increase intracellular molecular delivery of several different compounds. These studies show promise for the use of 3D printed acoustofluidic device for non-viral transfection of CRISPR/Cas9 in CAR-T cell processing.

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