Microfluidic purification of T lymphocytes separated from blood for chimeric antigen receptor T-cell manufacturing

Abstract

Background & Aim The success of CAR- T cell therapy for cancer immunotherapy is evidenced by near 90% remission rates for leukemic cancers and two CAR-T cell drugs have been approved by the FDA to date and commercialized. Despite these tremendous successes, the CAR T cell manufacturing process remains very complex and expensive hampered by major bottle necks that not only significantly reduce the cost effectiveness of the treatment but also impact its efficiency. Among these issues, the variability of the starting material for manufacture is important to consider. The percentage of purified and recovered T-cells separated from a patient's blood used for CAR T cell manufacture is highly variable due to many factors. The percentage of ‘non-wanted’ cells typically range anywhere from 10 to 90%, and these cells interfere with the downstream T-cell modification and expansion processes, potentially preventing the production of a high-quality end product. Microfluidic technologies, and more specifically inertial microfluidics which separate cells based on their physical attributes (e.g.size) has previously shown great success in separating cancer cells from blood but have not yet been applied for the separation of T-cells in the CAR T cell manufacturing process. This research thus aims to investigate the use of inertial microfluidics in the separation and purification of T-cells. Methods, Results & Conclusion We have designed several spiral microfluidic devices of various dimensions with enrichment and depletion outlets. We investigated the feasibility of using these devices to purify lymphocytes and more specifically T-cells from the leukocytes isolated from peripheral blood. We investigated the effect of channel dimensions, flow rate and microfluidic pressure on the separation of isolated leukocytes and determined the effect on cell viability and proliferation. The device was first optimized using a mixture of two cell lines differing in size; small Raji B lymphoblast-like cells and large A549 cells. We achieved 89 % recovery of the small Raji cells with a 95 % purity, while the majority of large cells were effectively depleted in the depletion outlet. Using leukocytes, we were able to achieve a 63 % (SD±0.04) recovery and 91 % (SD±0.06) purity for lymphocytes and a T-cell purity of 73 % (SD±0.1) from the whole leukocyte population. The viability and proliferation of the enriched lymphocyte cells remained unaffected in the separation process as measured by trypan blue staining (p