(M1130-06-31) Impact of Freezing Process Parameters on Jurkat Cell Cryopreservation

Purpose: Cryopreservation is the most widely used for long-term storage and transportation of cell therapy products. During freezing cells may lose cellular water with increased intracellular solute concentration. On the other hand, insufficient removal of intracellular water leads to intracellular ice formation, which can perturb structural integrity of the cells. A fine balance between Ice crystal formation and solute concentration is needed to prevent cell damage. Carefully controlling the cooling rate during freezing can be critical, while there are limited studies on the impact of controlled nucleation on intracellular solute concentration and subsequent ice formation. Controlled ice nucleation at a subzero temperature will lead to formation of extracellular ice and holding at this temperature would provide the cells with enough time to dehydrate, thus losing its intracellular water before freezing to lower temperatures. Impact of the ice nucleation temperatures and post nucleation hold time on cell cryopreservation has been investigated.

Methods: Jurkat cell, an immortalized line of human T lymphocyte cells was used to study the impact of the cooling process parameters on cell viability and metabolic activity. PlasmaLyte-A containing 10%,5% and 3%DMSO was used as the medium. A liquidous line was determined for all the formulations using differential scanning calorimetry (DSC). Controlled ice nucleation was introduced at two different temperatures: -6°C and -10°C and 10 minutes of post-nucleation holding time was used. A cooling rate of -1°C/min down to -80°C and warming rate of +10°C/min were used for the cooling and warming of the Jurkat cells(4-5*10⁶cells/ml). A Linkam-BCS196 temperature-controlled stage mounted on a fluorescence polarized microscope was used to study cellular volume changes and post freeze-thaw membrane integrity of the jurkat cells on a thin film platform. Cellular volume changes post nucleation and holding were determined from the captured time-lapse images using the Image-J software. Acridine orange and propidium iodide (AO/PI) were used to monitor membrane integrity of Jurkat cells post freeze-thaw. Furthermore, Jurkat cells were frozen in bulk in vials using a controlled rate freezer LyoStar III, following similar freezing profile. Thermocouples were added in vials to monitor product temperature. Cell titer glo assay was used to assess the metabolic activity of Jurkat cells post freeze-thaw. Results from these studies were compared with cells frozen without controlled nucleation.

Results: The liquidous line for the studied cryoformulations were generated by expressing the concentration as a function of the equilibrium freezing temperature. Liquidous line of the cryoformulations provides the unfrozen fraction of water and concentration of the maximal freeze-concentrate at a certain temperature. The concentration of maximal freeze-concentrate acts as the driving force for the osmotic dehydration of the cells. Time-lapse images taken from the microscope showed inducing ice nucleation and post nucleation hold time helps losing intracellular water of jurkat cells in 3% DMSO PlasmaLyte-A. Cellular volume changes measurement showed more efficient shrinking of the Jurkat cells post controlled nucleation when compared to cells frozen in spontaneous manner. During the freezing process (at the ice nucleation temperature), as the ice matures the concentration of the freeze concentrate increases along with the concentration of the DMSO in the freeze-concentrate. The increased freeze-concentrate concentration osmotically draws water out of the cell and drives membrane permeable coformulation components such as DMSO into the cell. Cell viability assessment showed that controlled ice nucleation and post nucleation hold time can help in achieving better viability of jurkat cells in 3% DMSO PlasmaLyte-A.

Conclusion: The information obtained from the liquidous line can be used for optimizing cooling parameters for Jurkat cells in studied cryoformulations. Optimizing the cooling protocol may reduce the concentrations of DMSO in the cryoformulations for effective cryopreservation of Jurkat cells.

Acknowledgements: Authors would like to acknowledge Janssen Pharmaceuticals for funding support.