A microfluidic chip for high throughput loading of cryoprotective agents into therapeutic allogeneic immune cells

Project Summary Allogeneic cell therapies offer transformative potential in clinical medicine, providing universal treatments with donor-derived immune cells and mitigating the variability of autogenic therapies. Among immune cell types, natural killer (NK) cells and neutrophils stand out for their therapeutic potential. NK cells can effectively target tumors and virally infected cells without patient-specific matching, while neutrophils, which do not require ABO matching, show promise in treating infectious diseases and inflammatory conditions. However, widespread adoption of allogeneic therapies is limited by challenges in cell cryopreservation, as current methods often compromise cell viability and function due to osmotic stress and toxicity during CPA loading. To address these limitations, we propose veloporation, a novel mechanoporation technique leveraging viscoelastic stretching to enable rapid, high-throughput CPA loading while minimizing toxicity. Unlike traditional CPA-loading methods, which expose cells to two primary injury mechanisms—molecular toxicity from prolonged CPA exposure and osmotic stress from excessive volumetric excursions—veloporation is the first approach to load CPAs almost instantaneously without altering cell volume. By alleviating both injury mechanisms simultaneously, veloporation preserves immune cell phenotype and functionality post-thaw, offering a transformative solution to the critical barrier of effective cell storage. The goal of this project is to develop and validate veloporation for CPA loading (velCPA) into immune cells, with a focus on NK cells and neutrophils for use in adoptive cell therapy (ACT). Aim 1 develops and optimizes the veloporation device for small immune cells. Aim 2 evaluates the ability of veloporation to load membrane-permeable and membrane-impermeable CPAs, minimizing toxicity and maximizing cell viability compared to traditional methods. Aim 3 assesses the post-thaw functionality of veloporated cells, including viability, immune activity, and potential for CPA co-delivery with therapeutic agents. By advancing cryopreservation techniques, this project addresses a critical barrier to the scalability of allogeneic cell therapies. It has the potential to enable the development of stable, off-the-shelf immune cell products for diverse clinical applications. While here we focus on NK cells and neutrophils, this technology could be adapted for the cryopreservation of other cell types used in regenerative medicine and immunotherapy, such as T cells or stem cells. The versatility and scalability of veloporation make it a promising platform technology with far-reaching implications for cellular therapies and biobanking.