Comparison of rigid polymer vials and flexible bags for cryopreservation of T cells

Abstract

Background & Aim The commercialization of cells for therapeutic use drives a critical need for the refinement of storage and handling procedures to ensure the integrity of cell-based products is maintained from manufacture through until administration to the patient. Autologous cell therapy applications currently call for large volume cell suspensions administered via intravenous injection, making cryogenic flexible bags an attractive containment solution. However, numerous challenges exist when using bags made from materials such as ethylene vinyl acetate (EVA) including filling difficulties, cell product loss due to dead volume, and cracking of the flexible material during the freeze/thaw process. Cyclic olefin polymer (COP) vials with rubber stopper-aluminum seal closures have desirable container closure integrity and material properties for cell therapy applications and have been shown to be compatible with numerous cell types in small volume (2 mL) format. This work evaluates the performance of larger 50 mL COP vials as an alternative for cryogenic storage of T cells. Methods, Results & Conclusion Peripheral blood mononuclear cells were isolated from healthy human donor blood and T cells were subsequently selected, stimulated, and expanded for 7 days. The T cells were then placed into either flexible EVA bags or rigid COP vials to be frozen for storage using a well-controlled process. The containers were thawed and the cells were cultured for a further 7 days. Cell growth, cell viability, T cell subsets, and exhaustion markers were evaluated between the first expansion and freezing, to establish the baseline. Container cryopreservation performance was determined by evaluating the same readouts immediately post-thaw and after the second expansion. Results indicate that the freezing and expansion processes may affect T cell subsets and exhaustion. The T cell subsets, exhaustion, and all other readouts examined revealed comparable results whether the cells were stored in a flexible bag or a rigid vial despite differences in cross-sectional area and time required to thaw. The findings highlight the value of understanding a cell-based product in the context of its handling and storage. Furthermore, the study has shown that 50 mL vials can provide a viable alternative for final containment of cell suspensions at low-temperature.