UC Riverside

Cancer immunotherapies using adoptive cell transfer (ACT) train a patient’s immune system to fight against cancer. This growing field of research has demonstrated promising results as an alternative treatment to current therapies, such as chemotherapy or radiation. However, the methods used to genetically engineer the anti-cancer functionalities into the required cell types (T lymphocytes), such as viral vector transduction, possess certain drawbacks that limit them in scalability, automation, and cost. There is a critical need for the development of a new cell modification strategy that addresses these shortcomings. We have looked towards the application of non-biological delivery methods as a potential means of addressing these limitations, namely microinjection.

Microinjection physically creates a transient membrane pore for the delivery of exogenous payloads. The gold standard of this technology consists of a human operator porating and injecting an exogenous payload through a linear “one-cell-at-a-time” scheme. This results in throughputs of ~3 cells/min, a rate that cannot feasibly produce the required number of treated cells for immunotherapies at an appreciable time scale. Thus, though this method has the advantage of safety and precision, it requires significant improvement to be competitive against current techniques for T-cell genetic modification.

We outline preliminary efforts to develop a new form of microinjection using silicon microfabrication. To simplify fabrication procedures, and demonstrate a proof-of- concept technology, we have created a device for ultrahigh (UHT) cellular manipulation via mechanical membrane poration, i.e. UHT mechanoporation. Such a device represents an interim step towards our microinjection concept.

The fundamental nature of the device stems from a microelectromechanical systems (MEMS) functional core composed of cell capture sites with monolithically integrated, sub-micrometer scale solid penetrators. Negative flow through aspiration vias at the bottom of the capture sites pulls cells onto the penetrators thus causing membrane poration. Cells are then released by reversing flow through the aspiration vias. The transient nature of membrane disruption enables transfection via diffusion-driven influx of exogenous molecules from the surrounding suspension, while massive parallelization provides for UHT operation (e.g. 10k capture sites in the current device).

We report successful fabrication of a first-generation UHT mechanoporation device capable of multiplexed cell manipulation. However, the preliminary studies reported by our collaborators indicated low cell delivery efficiencies (8%). We discuss our efforts to further optimize the operational parameters associated with our mechanoporation device. Through detailed device characterization, and implementation of precise, high-resolution experimental techniques, we have increased our device efficiencies by 10-fold. The delivery efficiencies for recovered, treated cells at the 30-minute time point and 12-hour time point were 90% and 97% respectively. Our overall device yields were 96% at the 12- hour time point, far higher than those reported by competing techniques, such as electroporation.

The results from our optimization studies demonstrate effective molecular delivery to cells using the UHT mechanoporation device. They indicate high delivery efficiencies across our parameter window. However, we observed a low efficiency of cell recovery from the device. Our results thus identify directions for future development of the UHT mechanoporation device, and collectively demonstrate the feasibility of an active injection system that can address limitations in engineered T-cell production.