Microfluidic mechanoporation for cell therapy

Advanced therapeutic methods, such as CAR-T cell anti-cancer immunotherapy, increasingly employ genetic modification of the patient's autologous cells, followed by their ex vivo expansion and re-infusion back into the patient. Good control of cell manipulation and adherence with the highest standards of good manufacturing practices are therefore of paramount importance throughout the therapy. Conventional methods for cell therapy, such as viral gene transfer, electroporation-based transfection or lipofection, affect cells in bulk, which cannot ensure the same transfection conditions for all cells, and limits cell viability, transfection efficiency and uniformity. In addition, they are often technically challenging, labor intensive, or consume a lot of expensive reagents. To overcome these limitations and ensure equal and reproducible transfection conditions at the single cell level, several microfluidic approaches have been proposed. Microfluidic mechanoporation delivers cargo into cells through membrane pores created by mechanical forces exerted on cell membrane in a microfluidic system, e.g., by squeezing the cells through narrow channels. However, before mechanoporation can be accepted for cell therapy, several technological and fundamental issues need to be addressed. For example, the underlying mechanisms of mechanoporation are not well understood and its biological effects on immune cells have not yet been explored. In this project, we will develop an innovative microfluidic system for efficient mechanoporation and transfection of immune cells that will solve some of the major problems of conventional methods. The system will combine mechanoporation with real-time optical visualization of cells and deformability cytometry. It will be designed for robust operation and efficient and precise reagent delivery. Using the system, we will be able to answer some fundamental open questions, such as how the state of the cell (e.g., size, deformability, properties of the cellular membrane and cytoskeleton) and the experimental parameters (e.g., temperature, shear stress) affect transfection efficiency and viability; how large the membrane pores are; how many plasmids need to enter the cell for successful transfection, etc. After poration, we will not only test the viability of the cells but also their physiological fitness and the speed of recovery. Understanding the mechanisms behind mechanoporation will significantly accelerate the optimization of experimental parameters for efficient transfection of immune cells, thus bringing the method closer to clinical application. In addition, the real-time feedback on the state of the cells will be critical for adherence to good manufacturing practices in the future. To achieve the proposed goal, the project will engage an interdisciplinary team from three groups with complementary expertise. The biophysical part will be performed by the Institute of Biophysics at the Faculty of Medicine at the University of Ljubljana, which comprises experts in theoretical and experimental biophysics of cell mechanics; they have recently developed a new method for assessing cell stiffness with optical tweezers and have all the necessary know-how for manipulating single cells in microfluidic systems. The tasks involving cell genetic manipulation and biomedical application will be carried out by the Department of Synthetic Biology and Immunology of the National Institute of Chemistry in Ljubljana, which has an outstanding track record in various applications of synthetic biology, genetic engineering and immunology. The design and fabrication of the microfluidic devices will draw on the expertise of the COMPETE Center at the University of Ljubljana, which is a multidisciplinary hub for microfluidic technologies funded by the EU Widespread scheme. The established infrastructure of COMPETE will be used also to disseminate the microfluidic technology to the public, industry and clinical users.