Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemical Engineering

Major Professor

Mark Jaroszeski, Ph.D.

Committee Member

Lawrence A. Stern, Ph.D.

Committee Member

Norma Alcantar, Ph.D.

Committee Member

Andrew Hoff, Ph.D.

Committee Member

Richard Heller, Ph.D.

Keywords

Electrotransfer, Atmospheric Plasma, Immunotherapy, Gene Delivery, Electroporation

Abstract

With gene-based immunotherapies on the rise to treat a multitude of diseases, the ability to genetically modify cells in a rapid and efficient fashion is necessary to keep up with demand. Gene electrotransfer (gene transfer by electroporation) is an appealing transfection method due to its low cost, high efficiency compared to other physical forms of gene delivery, and low toxicity compared to liposomal or viral vectors.

Traditional gene delivery via electroporation is done within a cuvette where the cell suspension is placed between two plate electrodes. This method requires cells to be moved from the treatment cuvette to a growth surface. The contact between the cells and two electrodes can cause significant cell damage and death. This motivated an investigation into an alternative delivery method utilizing an electric field that was applied by means of corona charge, a plasma formed in atmospheric air. This method utilizes one plate electrode and one needle electrode, where the needle electrode rests above the cell suspension rather than in contact with the suspension. This could potentially reduce, or even eliminate, the need to continuously move the cells from a treatment to growth surface, maintaining sterility. Ultimately, cells can be treated and grown in the same dish.

A novel system was designed in order to contain the cells and expose them to an electric field by utilizing corona charge to induce DNA uptake/electrotransfer to a human T cell line (Jurkat ATCC TIB-152). This system consisted of a custom dish with a metal bottom and non-conductive side walls. The metal bottom of the dish and a 28-gauge acupuncture needle were used to generate charge. There were many factors that impacted the creation of a corona charge such as the voltage, humidity, temperature, and distance between the needle and plate electrodes. These were investigated to identify parameters that could lead to successful transfection. It was determined that treatment time and electrical current (quantity of corona charge) had significant impact on the delivery of small molecules. These two parameters were varied to optimize delivery of small molecules and delivery while maintaining viability. Fluorescing cells were quantified using flow cytometry and fluorescence microscopy ~24-48 hours post treatment and viability was determined using DAPI. The goal was to maximize the number of GFP positive Jurkat cells while keeping viability as high as possible.

Treatment of the cells with corona charge treatment at high voltage caused an autofluorescence effect making it difficult to distinguish potential delivery of GFP encoding plasmid DNA. This led to alternative detection methods like luminescence and staining as well as the addition of an easier to transfect cell line, B16-F10. Plasmid encoding Luciferase-DDK-Myc was detected when the substrate luciferin comes into contact with the enzyme luciferase. Experiments showed that corona charge treatments caused a luminescence level that statistically significant in B16-F10 cells. Antibodies for both luciferase and GFP (anti-DDK, anti-GFP, and c-Myc (9E10) HRB) were utilized to stain the cells to confirm delivery with microscopy and flow cytometry.

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