NERDG 2026
Poster 29 Abstract
Impact of Freezing Parameters and Cryoformulation Design on Post-Thaw Viability of Jurkat Cells
Jobair Hossen (1), Nirnoy Dan (2), Sajal Patel (2), Peter Freed (3), Robin Bogner (1), Xiuling Lu (1)
(1) Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, USA; (2) AstraZeneca, Gaithersburg, Maryland, USA; (3) Roquette, Spring House, PA, USA
Presenting Author: Jobair Hossen
Corresponding Author: Xiuling Lu, [email protected]
Purpose
Cryopreservation is essential for the long-term storage and transportation of cell therapy drug products (CTDP). Current protocols often rely on uncontrolled ice nucleation, which can result in intracellular ice formation due to insufficient dehydration. CTDP formulations typically include 5–10% DMSO as a cryoprotectant; however, reducing DMSO content is desirable because of its associated toxicity. This work aims to investigate the effects of controlled ice nucleation, cryoformulation composition, and cooling rates on Jurkat cell cryopreservation. Furthermore, the relationship between predicted osmotic stress during freezing and post-thaw cell health was also examined.
Methods
Intracellular dehydration in Jurkat cells was assessed by cooling samples at different rates using a Linkam BCS196 cryostage mounted on a microscope. Time-lapse images were acquired during cooling, and cellular volumes were quantified with ImageJ to compare changes across cooling rates. Cells were frozen in 150 µL Linkam quartz crucibles with the BCS196 cryostage, and post-thaw viability was determined by AO/PI staining. To evaluate cryoprotective efficacy, various combinations of DMSO, glycerol, HSA, and trehalose in PlasmaLyte-A were tested for their ability to preserve post-thaw viability. For scaling up, Jurkat cells in 2 mL cryovials were frozen using a controlled-rate freezer, and cell health was subsequently assessed.
Results
Controlled ice nucleation at −6 °C significantly reduced the incidence of intracellular ice formation compared with uncontrolled nucleation. Following controlled nucleation, the average normalized cell volume at −24 °C was 0.18 ± 0.09, 0.20 ± 0.10, 0.25 ± 0.07, and 0.23 ± 0.05 across cooling rates of 0.5, 1, 5, and 10 °C/min, respectively. Corresponding average normalized cell viability values were 76.3 ± 2.7%, 76.4 ± 3.7%, 71.4 ± 8.5%, and 75.0 ± 6.9%. At the nucleation temperature, the ice-to-freeze-concentrate ratio was approximately 46:54.
Conclusion
Controlled ice nucleation significantly reduced intracellular ice formation compared with the uncontrolled group, and this reduction was associated with improved cell viability. Although changes in cell volume were observed at different cooling rates, these variations were not statistically significant. Post–freeze–thaw viability likewise showed no substantial differences among the experimental groups, suggesting that cooling rate had minimal influence on Jurkat cell dehydration under the tested conditions when intracellular ice formation was absent. Phase diagram analysis further enabled quantification of frozen and unfrozen fractions.
Keywords
Controlled ice nucleation, cryopreservation, cell therapy, cooling rate, intracellular ice
Poster 29 Abstract
Impact of Freezing Parameters and Cryoformulation Design on Post-Thaw Viability of Jurkat Cells
Jobair Hossen (1), Nirnoy Dan (2), Sajal Patel (2), Peter Freed (3), Robin Bogner (1), Xiuling Lu (1)
(1) Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, USA; (2) AstraZeneca, Gaithersburg, Maryland, USA; (3) Roquette, Spring House, PA, USA
Presenting Author: Jobair Hossen
Corresponding Author: Xiuling Lu, [email protected]
Purpose
Cryopreservation is essential for the long-term storage and transportation of cell therapy drug products (CTDP). Current protocols often rely on uncontrolled ice nucleation, which can result in intracellular ice formation due to insufficient dehydration. CTDP formulations typically include 5–10% DMSO as a cryoprotectant; however, reducing DMSO content is desirable because of its associated toxicity. This work aims to investigate the effects of controlled ice nucleation, cryoformulation composition, and cooling rates on Jurkat cell cryopreservation. Furthermore, the relationship between predicted osmotic stress during freezing and post-thaw cell health was also examined.
Methods
Intracellular dehydration in Jurkat cells was assessed by cooling samples at different rates using a Linkam BCS196 cryostage mounted on a microscope. Time-lapse images were acquired during cooling, and cellular volumes were quantified with ImageJ to compare changes across cooling rates. Cells were frozen in 150 µL Linkam quartz crucibles with the BCS196 cryostage, and post-thaw viability was determined by AO/PI staining. To evaluate cryoprotective efficacy, various combinations of DMSO, glycerol, HSA, and trehalose in PlasmaLyte-A were tested for their ability to preserve post-thaw viability. For scaling up, Jurkat cells in 2 mL cryovials were frozen using a controlled-rate freezer, and cell health was subsequently assessed.
Results
Controlled ice nucleation at −6 °C significantly reduced the incidence of intracellular ice formation compared with uncontrolled nucleation. Following controlled nucleation, the average normalized cell volume at −24 °C was 0.18 ± 0.09, 0.20 ± 0.10, 0.25 ± 0.07, and 0.23 ± 0.05 across cooling rates of 0.5, 1, 5, and 10 °C/min, respectively. Corresponding average normalized cell viability values were 76.3 ± 2.7%, 76.4 ± 3.7%, 71.4 ± 8.5%, and 75.0 ± 6.9%. At the nucleation temperature, the ice-to-freeze-concentrate ratio was approximately 46:54.
Conclusion
Controlled ice nucleation significantly reduced intracellular ice formation compared with the uncontrolled group, and this reduction was associated with improved cell viability. Although changes in cell volume were observed at different cooling rates, these variations were not statistically significant. Post–freeze–thaw viability likewise showed no substantial differences among the experimental groups, suggesting that cooling rate had minimal influence on Jurkat cell dehydration under the tested conditions when intracellular ice formation was absent. Phase diagram analysis further enabled quantification of frozen and unfrozen fractions.
Keywords
Controlled ice nucleation, cryopreservation, cell therapy, cooling rate, intracellular ice
