Poster V5 - Optimization of an Acoustic-Electric Shear Orbiting Poration Device for High Transfection Efficiency and Uniform Intracellular Delivery of mRNA.

Introduction: Efficient and uniform delivery of mRNA with maintained cell viability is imperative to immunotherapy, particularly in developing autologous chimeric antigen receptor T (CAR-T) cells with less chance of experiencing cytokine response syndrome (CRS). Our Acoustic-Electric Shear Orbiting Poration (AESOP) device utilizes Lateral Cavity Acoustic Transducer (LCAT) technology to trap cells in micro-vortices, caused by an oscillating air-liquid interface, and induce mechanical shear, generating nanopores in the cell membrane. Interdigitated gold electrodes patterned on glass introduce an electric field that expands these nanopores to deliver cargo larger than 20 kDa such as mRNA. Active mixing of cells and cargo in this format allows for intracellular delivery as cargo enters cells through the nanopores. To optimize our system for high transfection efficiency, uniform delivery, and viability of cells, we perform parametric testing of acoustic and electric field conditions.

Materials and

Methods: Intending to create universal CAR-T cells in the future, we use Jurkat cells as our model T cell and eGFP-mRNA as the cargo. Acoustic field parameter tests involve studying different-sized piezoelectric transducers and mixing times between electric field exposure. Electric field parameter tests include varying the voltage and amplitude, exposure time, and exposure intervals of the applied electric field. To assess transfection and uniformity, we obtained the percentage and coefficient of variation (CV%) of the eGFP profile of mRNA-transfected cells. We stained cells with Calcein AM Blue to confirm cell viability through active esterase activity in live cells.

Results, Conclusions, and Discussions: A PZT with a 40.5 kHz resonant frequency and an increased mixing time of 75 seconds improves the performance of our AESOP system. Three intervals of electric field exposure with 40 kHz frequency, 45 V amplitude, and 10 ms cycles are the ideal electric field parameters. Overall, AESOP offered superior outcomes with up to 80% transfection and > 85% viability compared to the gold standard of electroporation, the Lonza 4D nucleofection device, which provides an average transfection rate of 55% and 50% viability. Both devices maintain uniform transfection with CVs of ~57% for AESOP and 42% for Lonza, but the latter saturates cells, yielding higher median fluorescent intensities than AESOP. To confirm our optimal conditions translate to primary T cells, we will isolate CD3+ T cells from healthy patients and transfect them with mRNA-CAR using the new settings. Additional analysis of uniform delivery will be done with an antibody-bead assay to determine the number of CAR molecules on the surface of cells and a cytokine ELISA assay of cytokines common to CAR-T cell efficacy evaluation. Therefore, we show the AESOP device is a promising platform for producing CAR-T cells with higher transfection efficiency, uniformity of delivery, and viability of cells compared to electroporation.

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