Fan Yuan

T cell receptor (TCR) knockout is a critical step in producing universal chimeric antigen receptor T cells for cancer immunotherapy. A promising approach to achieving the knockout is to deliver the CRISPR/Cas9 system into cells using electrotransfer technology. However, clinical applications of the technology are currently limited by the low cell viability. In this study, we attempt to solve the problem by screening small molecule drugs with an immortalized human T cell line, Jurkat clone E6-1, for inhibition of apoptosis. The study identifies a few caspase inhibitors that could be used to simultaneously enhance the cell viability and the efficiency of plasmid DNA electrotransfer. Additionally, we show that the enhancement could be achieved through knockdown of caspase 3 expression in siRNA treated cells, suggesting that the cell death in electrotransfer experiments was caused mainly by caspase 3-dependent apoptosis. Finally, we investigated if the caspase inhibitors could improve TCR gene-editing with electrotransferred ribonucleoprotein, a complex of Cas9 protein and a T cell receptor-α constant (TRAC)-targeting single guide RNA (sgRNA). Our data showed that inhibition of caspases post electrotransfer could significantly increase cell viability without compromising the TCR disruption efficiency. These new findings can be used to improve non-viral T cell engineering.

<h4>Background</h4>Pulsed electric field has been widely used to facilitate molecular cargo transfer into cells. However, the cell viability is often decreased when trying to increase the electrotransfer efficiency. We hypothesize that the decrease is due to electropermeabilization of cell membrane that disrupts homeostasis of intracellular microenvironment. Thus, a reduction in the membrane permeabilization may increase the cell viability.<h4>Materials and methods</h4>Different compounds were supplemented into the pulsing buffer prior to electrotransfer for reduction of cell membrane damage. Extent of the damage was quantified by leakiness of the membrane to a fluorescent dye, calcein, preloaded into cells. At 24 hours post electrotransfer, cell viability and electrotransfer efficiency were quantified with flow cytometry.<h4>Results</h4>The cell viability could be substantially increased by supplementation of either type B gelatin or bovine serum albumin (BSA), without compromising the electrotransfer efficiency. The supplementation also decreased the amount of calcein leaking out of the cells, suggesting that the improvement in cell viability was due to the reduction in electrotransfer-induced membrane damage.<h4>Conclusion</h4>Data from the study demonstrate that type B gelatin and BSA can be used as inexpensive supplements for improving cell viability in electrotransfer.

Gallium nitride (GaN) is a compound semiconductor which has advantages to generate new functionalities and applications due to its piezoelectric, pyroelectric, and piezo-resistive properties. Recently, surface acoustic wave (SAW)-based acoustic tweezers were developed as an efficient and versatile tool to manipulate nano- and microparticles aiming for patterning, separating, and mixing biological and chemical components. Conventional piezoelectric materials to fabricate SAW devices such as lithium niobate suffer from its low thermal conductivity and incapability of fabricating multiphysical and integrated devices. This article piloted the development of a GaN-based acoustic tweezer (GaNAT) and its application in manipulating microparticles and biological cells. For the first time, the GaN SAW device was integrated with a microfluidic channel to form an acoustofluidic chip for biological applications. The GaNAT demonstrated its ability to work on high power (up to 10 W) with minimal cooling requirement while maintaining the device temperature below 32°C. Acoustofluidic modeling was successfully applied to numerically study and predict acoustic pressure field and particle trajectories within the GaNAT, which agree well with the experimental results on patterning polystyrene microspheres and two types of biological cells including fibroblast and renal tumor cells. The GaNAT allowed both cell types to maintain high viabilities of 84.5% and 92.1%, respectively.

Correction for 'Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB' by Roman Mikhaylov et al., Lab Chip, 2020, 20, 1807-1814, DOI: 10.1039/C9LC01192G.

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. In this paper, we develop a novel and simple technology to fabricate a surface acoustic wave (SAW)-based acoustofluidic device by clamping electrodes made using a printed circuit board (PCB) with a piezoelectric substrate. The PCB-based SAW (PCB-SAW) device is systematically characterised and benchmarked with a SAW device made using the conventional photolithography process with the same specifications. Microparticle manipulations such as streaming in droplets and patterning in microchannels were demonstrated in the PCB-SAW device. In addition, the PCB-SAW device was applied as an acoustic tweezer to pattern lung cancer cells to form three or four traces inside the microchannel in a controllable manner. Cell viability of ∼97% was achieved after acoustic manipulation using the PCB-SAW device, which proved its ability as a suitable tool for acoustophoretic applications.