Date on Master's Thesis/Doctoral Dissertation

5-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Interdisciplinary and Graduate Studies

Degree Program

Interdisciplinary Studies with a specialization in Bioinformatics, PhD

Committee Chair

Kopechek, Jonathan

Committee Co-Chair (if applicable)

Rousell, Thomas

Committee Member

Rousell, Thomas

Committee Member

Chen, Joseph

Committee Member

Bates, Paula

Committee Member

Yaddanapudi, Kavitha

Author's Keywords

T cells; Immunotherapy; acoustofluidics; microbubbles; ultrasound

Abstract

Cell-based therapies represent the latest biotechnological revolution in medicine, but significant limitations exist in non-viral intracellular delivery techniques during manufacturing of cell therapies. To address these limitations, a novel acoustofluidic device was developed to deliver ultrasound waves into a flow chamber for rapid molecular delivery to human cells. Our acoustofluidic device delivers biomolecules by inducing expansion and collapse of exogenous gas-filled microbubbles (“cavitation”), which enhances molecular delivery to nearby cells. Experimental studies were conducted to characterize key parameters that influence acoustofluidic-mediated molecular delivery to human cells. A clinical ultrasound transducer was generally utilized with varying cationic microbubble concentrations. Intracellular delivery of a fluorescent dye was assessed using flow cytometry. Preliminary studies with erythrocytes and primary T cells used a polydimethylsiloxane-based acoustofluidic device, while subsequent studies with Jurkat T cells used a 3D-printed acoustofluidic device to increase compatibility for cell manufacturing applications. For erythrocytes, ultrasound pressure and microbubble concentration were important parameters that influenced fluorescein delivery. Initial Jurkat T cell studies indicated that microbubble concentration, surface charge, and ultrasound pressure influenced molecular delivery. Subsequent experiments directly compared refined parameters of the acoustofluidic device and a static ultrasound configuration. The acoustofluidic device and the static ultrasound configuration had similar levels of intracellular delivery with small (calcein) and large (150 kDa-FITC Dextran) molecules, although less ultrasound exposure and lower microbubble concentrations were required in the acoustofluidic device. Bioeffects from acoustofluidic treatment were assessed in subsequent experiments. Cell plating density and media formulation influenced acoustofluidic-mediated molecular delivery. Confocal microscopy indicated that actin patches form in response to acoustofluidic treatment and plug perforations in the plasma membrane. In addition, disruption of organelle structures, such as the nucleus, were observed after acoustofluidic treatment. Cell cycle phases also influenced molecular delivery, and cell proliferation was briefly reduced after acoustofluidic treatment. The results of this dissertation indicate that acoustofluidic parameters and biological characteristics have important effects on molecular delivery to human cells. Our acoustofluidic device achieved similar molecular delivery levels to Jurkat T cells as a static ultrasound configuration, which indicate that acoustofluidic devices offer potential advantages for cell processing applications and may improve manufacturing of cell therapies.

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