NERDG 2026
Poster 38 Abstract
Impact of Cryoformulation Contact Time on Jurkat Cell Viability
Alice Han (1), Michael Tang (2), Jobair Hossen (3), Xiuling Lu (3)
(1) Edwin O. Smith High School, Storrs, CT 06268, USA (2) Northern Valley Regional High School at Old Tappan, Old Tappan, NJ 07675, USA (3) Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
Presenting and Corresponding Authors: Alice Han, [email protected] and Michael Tang , [email protected]
Purpose
Cell cryopreservation is an essential step in cell therapy, also used in a multitude of different biological product development. It aims to maintain biological activity, enabling long-term storage of cells for future treatment. Typically, cryopreservation formulations contain cryoprotectants, including cell-permeating agents such as dimethyl sulfoxide (DMSO) and glycerol, as well as non-cell-permeating agents such as human serum albumin (HSA) and trehalose. The cells will be exposed to these agents prior to freezing and after thawing. Long contact time can affect the viability of the cells. The current established protocol recommends a contact time of 1 hour to ensure minimal effect on cell viability. However, longer contact time is desired during the manufacturing process. This study therefore aims to evaluate the maximum contact time for the model T-cell (Jurkat cells) in various cryoprotectant formulations, including reduced DMSO content formulations intended to improve the safety of T-cell therapy.
Methods
To measure Jurkat cell viability, cells were mixed with Acridine Orange/Propidium Iodide (AO/PI) or Trypan Blue, then pipetted in a CellDrop Automated Cell Counter to be analyzed. The experiment was conducted for up to 360 minutes, with cell density and viability assessed at time points in increments of 60 minutes. Jurkat cells were incubated with various formulations of 2.5-10% DMSO, glycerol, HSA, and trehalose in PlasmaLyte-A.
Results
Cell viability declined with increasing cryoprotectant contact time, with a more noticeable decrease observed after 180 minutes, however it remained relatively high (70-80% viability) for formulations with 2.5% DMSO. Formulations with >5% DMSO after 180 minutes yielded 60-70% viability. It was found that assessments with Trypan Blue showed higher cell viability than AO/PI, suggesting that Trypan blue overestimated viability.
Conclusion
The overall effect of contact time on Jurkat cell viability varies greatly across different cryoformulations. The optimal formulation enables a 3 hour contact time with cells without significant decrease in cell viability. This formulation will as a result have the potential to improve cell cryopreservation outcome.
Keywords
Jurkat, Cryopreservation, Viability, Cryoformulation, DMSO
Poster 38 Abstract
Impact of Cryoformulation Contact Time on Jurkat Cell Viability
Alice Han (1), Michael Tang (2), Jobair Hossen (3), Xiuling Lu (3)
(1) Edwin O. Smith High School, Storrs, CT 06268, USA (2) Northern Valley Regional High School at Old Tappan, Old Tappan, NJ 07675, USA (3) Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
Presenting and Corresponding Authors: Alice Han, [email protected] and Michael Tang , [email protected]
Purpose
Cell cryopreservation is an essential step in cell therapy, also used in a multitude of different biological product development. It aims to maintain biological activity, enabling long-term storage of cells for future treatment. Typically, cryopreservation formulations contain cryoprotectants, including cell-permeating agents such as dimethyl sulfoxide (DMSO) and glycerol, as well as non-cell-permeating agents such as human serum albumin (HSA) and trehalose. The cells will be exposed to these agents prior to freezing and after thawing. Long contact time can affect the viability of the cells. The current established protocol recommends a contact time of 1 hour to ensure minimal effect on cell viability. However, longer contact time is desired during the manufacturing process. This study therefore aims to evaluate the maximum contact time for the model T-cell (Jurkat cells) in various cryoprotectant formulations, including reduced DMSO content formulations intended to improve the safety of T-cell therapy.
Methods
To measure Jurkat cell viability, cells were mixed with Acridine Orange/Propidium Iodide (AO/PI) or Trypan Blue, then pipetted in a CellDrop Automated Cell Counter to be analyzed. The experiment was conducted for up to 360 minutes, with cell density and viability assessed at time points in increments of 60 minutes. Jurkat cells were incubated with various formulations of 2.5-10% DMSO, glycerol, HSA, and trehalose in PlasmaLyte-A.
Results
Cell viability declined with increasing cryoprotectant contact time, with a more noticeable decrease observed after 180 minutes, however it remained relatively high (70-80% viability) for formulations with 2.5% DMSO. Formulations with >5% DMSO after 180 minutes yielded 60-70% viability. It was found that assessments with Trypan Blue showed higher cell viability than AO/PI, suggesting that Trypan blue overestimated viability.
Conclusion
The overall effect of contact time on Jurkat cell viability varies greatly across different cryoformulations. The optimal formulation enables a 3 hour contact time with cells without significant decrease in cell viability. This formulation will as a result have the potential to improve cell cryopreservation outcome.
Keywords
Jurkat, Cryopreservation, Viability, Cryoformulation, DMSO
