mRNA-based CAR T-cells manufactured by miniaturized two-step electroporation produce selective cytotoxicity toward target cancer cells

Abstract

There is a growing interest for viral vector-free chimeric antigen receptor (CAR) T-cells due to its ability to kill cancer cells without adverse side effects. A potential avenue for manufacturing...

Recent Advances in Microscale Electroporation

“…It takes 3–5 min to align a single cell, parallelization is challenging, and optical forces may not be sufficiently strong to easily move particularly sticky cells . Instead, DEP forces have been used frequently to position cells in microscale EP devices. ,,,,,− Unlike optical tweezers, DEP forces are effective for large numbers of cells in parallel. DEP forces are exerted on polarizable particles using a nonuniform electric field to manipulate cells at the microscale and have been widely used for microfluidic cell sorting. , The earliest example of DEP to assist microscale EP used triangular electrode arrays to draw cells toward sharp-edged electrodes in a microfluidic channel for yeast, bacteria, plant, and mammalian cell lysis at low voltages. , Kim et al used DEP forces to guide bacteria into a microwell array with single-cell trapping. − The DEP forces were stronger than bacteria motility to restrict cells from swimming out of the microwells.…”

“…Punjiya et al manipulated cell position by changing the flow rate and drag forces applied to cells in relation to DEP forces generated using half-ring and flat electrodes . In subcellular channel devices, DEP is a label-free positioning technique that can be applied by changing voltage pulses. ,, In other systems, dedicated DEP electrodes have been fabricated on-chip for single-cell loading over separate EP electrodes. , Jayasooriya et al used DEP forces to align large numbers of primary T cells along the gap between interdigitated electrodes to apply more uniform conditions across all samples . Magnetic forces can also be applied to reposition cells conjugated with magnetic beads. , Using patterned, on-chip magnetic features, magnetic tweezers may be formed for parallelized loading of cells onto static cell arrays (Figure D) .…”

“…High-throughput microscale EP systems are also applicable to program immune cells, potentially for reintroduction into patients to target diseased cells. ,,, Select studies have delivered GFP-encoding mRNA into primary human T cells at up to 2 cells/min to mimic potential cell engineering for adoptive cell transfer. , An emergent cancer therapeutic strategy is the engineering of synthetic chimeric antigen receptors (CARs) to program immune cells to target cells expressing specific antigens . Chang et al transfected NK cells with CAR-encoding plasmids as an early demonstration of immune cell engineering using microfluidics .…”

“…Chang et al transfected NK cells with CAR-encoding plasmids as an early demonstration of immune cell engineering using microfluidics . Jayasooriya et al characterized the cellular response to CAR-encoding mRNA into primary T cells using interdigitated electrodes . GFP and CAR levels peaked 24 and 48 h after mRNA delivery, respectively, underscoring the differences in gene expression based on mRNA sequences.…”

“…188,200 Jayasooriya et al used DEP forces to align large numbers of primary T cells along the gap between interdigitated electrodes to apply more uniform conditions across all samples. 201 Magnetic forces can also be applied to reposition cells conjugated with magnetic beads. 146,202 Using patterned, on-chip magnetic features, magnetic tweezers may be formed for parallelized loading of cells onto static cell arrays (Figure 7D).…”