Ultrasound mediated cell transfection in microchamber systems

Home Institution: Boston University NNCI Site: KY Multiscale @ University of Louisville REU Principal Investigator: Kevin M. Walsh Ph.D. - Dept. of Electrical and Computer Engineering, University of Louisville REU Mentor: Jonathan Kopechek - Dept. of Bioengineering, University of Louisville Abstract: In the realm of emerging cancer therapies, chimeric antigen receptor (CAR)-T cell therapy stands out for its potential to revolutionize cancer treatment. By harnessing and reprogramming a patient's white blood cells to target cancer cells, this therapy promises precision and efficacy against even the most resistant cancers. However, a current barrier with CAR-T cell therapy is the time spent genetically modifying T-cells in the lab. The Kopechek lab aims to address this by optimizing cell transfection, or the artificial insertion of cancer-fighting genes into T-cells. Our approach utilizes ultrasound-driven microbubble oscillation to deliver genetic material into cells. Furthermore, we employ polydimethylsiloxane (PDMS) microchamber wells fabricated from an SU8 mold for detailed single-cell analysis of the transfection process. In our study, we used bright field, phase contrast, and fluorescence microscopy to analyze Jurkat T-cells seeded on microchamber wells coated with fibronectin. We identified an optimal well size of 50 microns, a seeding cell concentration of 50,000 cells/ml, and an ultrasound treatment time of one day after cell seeding. We also identified methods to reduce air bubbles in cell media due to PDMS aeration. Finally, we imaged samples over time to track cell migration and proliferation, and we created a MATLAB-based computational analysis method to analyze fluorescence-stitched microscopy images. These results hold the potential to enhance the precision and efficiency of cell seeding in microchamber wells and set the stage for in-depth observation and machine-learning analyses of the transfection process.