(161e) A Multilayered Approach to Continuous Freezing Process Design for Human Induced Pluripotent Stem Cells

Human induced pluripotent stem (hiPS) cells are one of the most promising sources in the field of regenerative medicine because the cells have various advantages over the conventional sources such as human embryonic stem cells. Along with recent successful clinical studies, e.g., Parkinson’s disease and retinitis pigmentosa, the implementation of medical treatments using hiPS cells is in progress.

In hiPS cell manufacturing, freezing processes are one of the most critical steps because the process is needed to the transportation and preservation of the cells. Generally, two methods can be adopted for the freezing process of hiPS cells: slow freezing and vitrification. In slow freezing, cryovials filled with cell suspension are cooled in a programmed freezer at a predetermined cool rate. In vitrification, the vials are immediately cooled using liquid nitrogen. At a commercial scale, vitrification is rarely adopted in the cell therapy industry because of the scale limitation, process complexity, and very high concentrations of cryoprotective agents. Therefore, in this work, we focus on the slow freezing option.

In general, the slow freezing of hiPS cells has been performed by a direct contact freezer that can accommodate only a limited number of cryovials. On the other hand, with the expected future commercialization, there is a need to design continuous slow freezing processes for hiPS cells that can handle many cryovials. In the field of process systems engineering, several regenerative medicine-related processes have been explored by model-based approaches, e.g., fed-batch mammalian cell culture¹, supply chain of autologous chimeric antigen receptor T-cell therapy², and batch freezing of hiPS cells³. However, process design of continuous cell freezing is yet to be performed.

This work presents a multilayered approach to continuous slow freezing process design for hiPS cells. From a process modeling perspective, it is necessary to understand the connection between the cell, cryovial, and freezer layers to model the continuous freezing process for hiPS cells. We developed a model to cover the cell, cryovial, and freezer layers. Given the conveyor belt velocity and the inlet coolant velocity in a freezer, the developed model can calculate the cell survival rate after thawing and the required freezing time.

The application of the developed model was demonstrated in a case study. As a result, an optimal set of operating conditions was obtained for the multiple objective functions, e.g., cell survival rate. These results would be useful for designing continuous freezing processes for hiPS cells. In the ongoing work, we are investigating different sizes of freezers, and also planning experimental investigation of the calculated results.

References

1. Gan J, Parulekar SJ, Cinar A. Development of a recursive time series model for fed-batch mammalian cell culture. Comput Chem Eng. 2018;109:289-298.

2. Papathanasiou MM, Stamatis C, Lakelin M, Farid S, Titchener-Hooker N, Shah N. Autologous CAR T-cell therapies supply chain: challenges and opportunities? Cancer Gene Ther. 2020;27:799-809.

3. Hayashi Y, Horiguchi I, Kino-oka M, Sugiyama H. Model-based assessment of temperature profiles in slow freezing for human induced pluripotent stem cells. Comput Chem Eng. 2021;144:107150.