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Science

Current cancer cell therapies often rely on modified immune cells that are programmed with new “instructions” to recognise and kill tumour cells, but today this is usually done using viruses, which are expensive, slow to produce and can raise safety concerns. In this work, we developed a small fluidic device that gently squeezes cells through tiny holes so that genetic material in the form of mRNA can slip inside without using any virus or toxic chemicals. The treated cells then briefly produce special receptors, called CARs, on their surface, allowing them to detect cancer markers and release warning and killing signals such as cytokines when they meet the right target. Because this process is fast, uses standard cell manufacturing systems, and can handle very large numbers of cells in minutes, it could help make advanced cell therapies more accessible to patients.

Societal Impact

Cell therapies like CAR T have transformed outcomes for some blood cancers, but their use is limited by long manufacturing times and very high production costs driven largely by viral vectors, specialised equipment, and long production times. Our mechanically based, non viral method uses a simple inline device and serum free media to process roughly 100 million cells per square centimetre of membrane area at more than 100 million cells per minute, reducing dependence on bespoke viral manufacturing facilities. By enabling transient mRNA based engineering of patient T cells at scale, this approach could lower per dose manufacturing costs, shorten turnaround from weeks to days, and improve global capacity for both autologous and future allogeneic products, thereby broadening patient access while easing infrastructure and workforce demands. The technology is also versatile enough to deliver other biomolecules, supporting innovation in next generation immune and regenerative cell therapies that could further benefit public health.

Technical Summary

We report a 3D printed, injection moldable inline mechanoporation cartridge that incorporates track etched polycarbonate membranes (10–12 μm pores) within a luer lock housing and is driven by conventional peristaltic pumps, enabling closed system processing compatible with existing clinical cell manufacturing platforms. Jurkat cells were used to map key parameters, including cell–pore size ratio, flow rate, and payload concentration, showing that >35% dextran uptake with >60% recovery at a loading of roughly 100 million cells per cm² of membrane can be routinely achieved. Transfection efficiency increased with flow rate and with progressive pore occlusion (fraction collection experiments), consistent with elevated local shear, while increasing mRNA concentration mainly boosted per cell expression (MFI) rather than the fraction of eGFP positive cells, implying an intrinsically non responsive subpopulation.

Activated human PBMCs was more refractory to mechanoporation than Jurkat, with eGFP mRNA transfection efficiencies of ≈12.5% versus ≈30% at comparable conditions. Anti CD19 CAR mRNA (2,434 nt) delivery achieved 15–17% CAR⁺ T cells, which demonstrated antigen specific IFN γ and TNF α secretion upon CD19 engagement, confirming functional transient CAR T generation. Transcriptomic profiling of exhaustion and activation markers suggested that passage through the device partially resets activated T cells toward a less exhausted state without excessive loss of viability, and simple scaling by membrane area maintained overall transfection efficiency across 5–200 million cell runs, supporting straightforward translation toward autologous and allogeneic manufacturing.

Figure 1. Mechanical stresses transiently permeabilize cell membranes, allowing different payloads to enter the cells. These payloads can include genetic material such as mRNA, and result in transient expression of genes such as chimeric antigen receptors (CAR) on the cell surface, enabling the cells to recognize and kill target tumor cells.

References

Non‐Viral Manufacturing of Cellular Immunotherapy Using Simple Mechanical Transfection Device Y Lee, W Wong, T Seah, D Yew, CW Beh, Advanced Therapeutics 8 (10), e00094 https://advanced.onlinelibrary.wiley.com/doi/abs/10.1002/adtp.202500094