(M1230-09-55) Impact of Freezing Process and Trehalose Concentrations on T-Cell Cryopreservation

Purpose: Cryopreservation is commonly used for the long-term storage and transportation of cell therapy drug products (CTDPs). Most existing protocols rely on uncontrolled ice nucleation, although some utilize controlled ice nucleation. However, the effects of different freezing methods, particularly uncontrolled versus controlled ice nucleation, on intracellular dehydration and overall cryopreservation outcomes remain poorly understood. CTDPs typically contain 7.5 to 10% DMSO, but increasing attention is being given to DMSO-free formulations or to reducing DMSO concentrations to mitigate associated side effects. This study aims to evaluate the effects of controlled ice nucleation and freezing parameters on Jurkat cell cryopreservation using PlasmaLyte A and trehalose. Intracellular trehalose delivery via incubation was investigated as a strategy to reduce DMSO dependence while preserving post-thaw viability and membrane integrity. Additionally, the relationship between model-predicted osmotic stress during extracellular freezing and post-thaw cell health was assessed to better understand the role of freezing rate and cell dehydration in cryopreservation.

Methods: To measure intracellular dehydration, Jurkat cells were cooled at three different rates: 0.5 °C/min, 1 °C/min, and 2 °C/min, using a Linkam BCS196 cryostage mounted on a microscope. Controlled ice nucleation was compared with uncontrolled ice nucleation. Time-lapse images were captured during the cooling process and analyzed using ImageJ to calculate cellular volume. Trehalose uptake was quantified using the Megazyme trehalose assay kit, following the manufacturer’s instructions. Jurkat cells (6 × 10⁶ cells/mL) were incubated with 3.5%, 7%, 10%, or 14% (w/v) trehalose in culture media. After incubation, 3 × 10⁶ viable cells were collected, centrifuged at 10,000 rpm for 10 minutes, washed twice with PlasmaLyte A to remove extracellular trehalose, and lysed in 0.05% Triton X/PlasmaLyte A. Cell viability was measured using acridine orange/propidium iodide (AO/PI) staining. Apoptosis was assessed by staining 5 × 10⁵ cells with Annexin V, Hoechst, and propidium iodide in Annexin V binding buffer. Cells were incubated at 37 °C for 15 minutes, then washed and pelleted before analysis. Flow cytometry gating of the fluorescent signals was used to identify and quantify the cell populations.

Results: With uncontrolled ice nucleation, cell volume decreased by 15.40 ± 12% at 0.5 °C/min, 15.21 ± 13.67% at 1 °C/min, and 12.82 ± 17.17% at 2 °C/min. In contrast, controlled nucleation at –6 °C led to significantly greater volume decreases of 89.56 ± 5.1%, 79.6 ± 8.12%, and 84.25 ± 7.89% at cooling rates of 0.5 °C/min, 1 °C/min, and 2 °C/min, respectively. Intracellular trehalose uptake by Jurkat cells increased with time and extracellular concentration (Figure 1). At 3 hours, intracellular trehalose levels were 1.1 ± 0.2 mg/mL and 3.4 ± 0.4 mg/mL with 3.5% and 7% (w/v) extracellular trehalose, respectively. After 6 hours, these increased to 3.5 ± 0.2 mg/mL and 8.6 ± 0.4 mg/mL, with the highest uptake of 11.4 ± 0.7 mg/mL observed at 14% (w/v) extracellular trehalose. Cell viability after 6 hours of trehalose incubation was approximately 84%, 80%, and 70% at 7%, 10%, and 14% trehalose, respectively. Metabolic viability decreased to about 40% at 7% and 56% at 14% trehalose. Proline at 50 mM concentration improved metabolic viability from ~65% to ~80% at 24 hours in 7% and 10% trehalose. Apoptosis increased with trehalose concentration, with 10% causing a 10% rise in late apoptosis and 25% rise in early apoptosis.

Conclusion: Controlled ice nucleation substantially enhanced intracellular dehydration in Jurkat cells compared to uncontrolled nucleation, indicating more effective water efflux during freezing. While some variation in dehydration was observed across cooling rates under controlled nucleation, the differences were not significant. Controlled ice nucleation resulted in more homogeneous cell volume compared to uncontrolled nucleation. Intracellular trehalose uptake was found to be both concentration- and time-dependent. The addition of proline to the trehalose-containing media enhanced cellular metabolic viability under high osmotic stress induced by extracellular trehalose and provided protection by reducing apoptosis.

Acknowledgements: We gratefully acknowledge the financial support from Center for Pharmaceutical Processing Research (CPPR) and University of Connecticut for this work. Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.