Chimeric Antigen Receptor (CAR) T-cell therapy offers life-saving potential, particularly against blood cancers, but severe side effects such as cytokine release syndrome (CRS) limit its safety. These toxicities are linked to uncontrolled CAR expression levels on the T-cell surface. Led by Professor Abraham P. Lee, researchers at the University of California, Irvine, have developed an advanced microfluidic system, called the Acoustic-Electric Shear Orbiting Poration (AESOP) platform, to precisely control the dose of genetic material delivered into primary T cells. This innovation promises safer, more homogeneous, and highly effective cellular immunotherapies. 

The Urgent Need for Fine Control

CAR T-cell therapy is a revolutionary advancement in cancer treatment, demonstrating unprecedented efficacy, especially for haematologic malignancies like B-cell acute lymphoblastic leukaemia (B-ALL). Despite this remarkable success, the therapy can induce potentially life-threatening toxicities, most notoriously CRS, where excessive inflammatory cytokines are released, leading to systemic immune activation. Crucially, studies have revealed that the heterogeneity and high density of CAR expression on the surface of T cells often determines both efficacy and safety, as T cells expressing a high density of CAR molecules (CAR^High T cells) are linked to excessive cytokine release and exhausted phenotypes, increasing the potential for CRS. Conversely, CAR^Low T cells may not be potent enough to interact effectively with tumour cells. Therefore, it’s necessary to achieve a precisely controlled method to manufacture CAR T cells with CAR expression density titrated within an optimal range.

Traditional manufacturing methods often fall short of this precision. Clinical settings frequently rely on viral vectors (such as retroviruses and lentiviruses), which offer high transduction efficiency but carry severe safety concerns, including insertional oncogenesis and heterogeneity in gene expression. Alternatively, bulk electroporation (a non-viral method that uses high voltages to temporarily destabilise the cell membrane) can deliver genes, but the high voltages often compromise cell viability and function, perturbing gene expression and cytokine production. Moreover, bulk methods lack the mixing process necessary to achieve uniform and dosage-controlled delivery across the entire cell population

Building a Better ‘Bio-CPU’

To overcome these limitations, Prof Lee and colleagues developed the Acoustic-Electric Shear Orbiting Poration (AESOP) platform. This technology acts as a ‘biological cell processing unit’ (bio-CPU) analogous to a CPU in computing, but for bioengineering.

The AESOP device uses a two-step membrane disruption strategy, combining gentle mechanical shear with a low-intensity electric field, which enables the uniform delivery of genetic cargo into large populations of cells. The core innovation lies in the use of lateral cavity acoustic transducer (LCAT) technology, which traps cells inside acoustic microstreaming vortices.

The AESOP process consists of 3 stages:

1. Shear-Induced Poration – Cells are trapped in whirlpool-like microstreaming vortices and subjected to oscillatory mechanical shear stress near oscillating air–liquid interfaces. This gentle, tunable, and uniform mechanical shear opens nano-sized pores on the cell membranes. The process is safe, as low to moderate shear forces (LS and MS) do not disrupt the function of primary T cells, unlike the substantial increase in pro-inflammatory cytokines (IFN-γ and TNF-α) observed after traditional electroporation.
2. Electric Field Modulation – Once nanopores are present, a gentle, low-strength electric field is applied via integrated electrodes. This field uniformly enlarges the pre-existing pores without relying on the high, damaging voltages required by bulk electroporation. This two-step strategy maintains cell viability, preserving primary T-cell genotype and minimising cytokine release function.
3. Chaotic Mixing and Uniform Uptake – The microstreaming vortices are crucial for achieving dosage control. They generate chaotic mixing, ensuring that the genetic material (cargo) is uniformly mixed with the T cells. As a result, each cell takes up approximately the same dosage of cargo.

Precision Pumping and Handling Large Cargo

The AESOP platform demonstrates significant advantages over conventional methods in terms of efficiency, viability, and homogeneity. Prof Lee showed that AESOP achieved a delivery efficiency of 76±9.76% and cell viability of 80.4±5.48% for primary T cells, significantly outperforming bulk electroporation, which yielded 47.5±12.74% efficiency and 44.93±8.29% viability.

Furthermore, the original AESOP design proved highly effective at handling large genetic cargos. AESOP achieved high delivery efficiency (>90%) and high cell viability (>80%) across a wide range of molecules, from small dyes (<1 kDa) up to large 2 MDa dextran molecules, in a variety of cell lines.

This capability to deliver substantial genetic material is critical for modern gene editing. For instance, AESOP successfully delivered large plasmids for eGFP (enhanced green fluorescent protein) expression (6.1 kbp) and even larger plasmids (9.3 kbp) encoding the CRISPR-Cas9 system for targeted gene knockout. The latter is challenging, as these cargo sizes often exceed the packaging capacity of popular viral vectors like adeno-associated viruses (AAVs).

A key metric demonstrating AESOP’s precision is the coefficient of variation (%CV), which quantifies the uniformity of cargo uptake across the cell population. A lower %CV indicates a more monodisperse (uniform) dosage taken by the cells. While bulk electroporation resulted in a high %CV of 97% for dextran delivery, AESOP reduced this dramatically to approximately 53.6%. Similarly, when delivering plasmid DNA, AESOP maintained a consistent and low %CV around 50%, compared to control groups with %CV >120%, showing greater control and consistency in delivery.

Titrating T-Cell Expression for Safety

The AESOP platform’s greatest functional advantage is its ability to precisely control the dosage delivered. The researchers successfully demonstrated this capability by showing a linear relationship between the input concentration of reagents (dextran and mRNA) and the resulting fluorescence intensity within the cells. For dextran, fluorescence was linearly correlated to concentration in the AESOP groups (R²=0.99), but this did not hold true for the electroporation groups (R²=0.83).

Applying this precision to CAR T-cell generation, the team used mRNA encoding anti-CD19 and a GFP-reporter. Using mRNA instead of plasmids or viral vectors allows for the transient expression of CAR, avoiding the permanent genomic integration and potential long-term risks associated with permanent expression systems (like B-cell aplasia or on-target off-tumour toxicity).

The results showed that AESOP could control the expression levels of the CAR protein by varying the input mRNA dosage (2.5, 5.0, and 7.5 μg). As the dosage of CAR mRNA increased, the CAR expression level, measured by fluorescence intensity, also increased. This finding is critical because it demonstrates the ability to titrate the CAR density on the T-cell surface, allowing researchers to potentially create CAR^High, CAR^Medium, and CAR^Low T cells. Crucially, the electroporation control group failed to show any observable shift in fluorescence intensity peaks as the mRNA dosage increased; expression levels were mostly saturated, or simply widespread and uncontrolled.

Scaling into the Future

The success of CAR T-cell therapy in a clinical setting demands platforms capable of high-throughput cell processing, often requiring millions to billions of transduced T cells.

The AESOP platform was modified to increase its throughput by modifying the system from a serial channel design to seven parallel microfluidic channels and increasing the channel height from 40 μm to 60 μm. The enhanced throughput allows the system to efficiently process the necessary volume of cells for clinical adoption. Prototypes processing up to a billion cells per chip are being developed, while still maintaining the control of dosage per cell. Commercialization efforts for these are underway by a startup called CellEcho Biotech.

By ensuring uniform, dosage-controlled, and high-throughput delivery, the AESOP acoustic-electric microfluidic system is a significant step forward in cell therapy manufacturing. The platform’s precision addresses the toxicities linked to uncontrolled CAR expression, providing a well-controlled process for generating uniform CAR T cells. Moreover, for more complex cell engineering applications the AESOP is capable of delivering multiple cargos with precise dosage. As such, this platform lays the foundation for future bio-CPUs, allowing the precision processing and ‘programming’ of cells with clinically relevant throughput. This ability to precisely titrate CAR expression levels will be vital for future scientific studies investigating how CAR density affects T-cell functionality, such as cytotoxicity and cytokine release, in physiological environments.