(157g) Model-Based Assessment of Design Parameters in Freezing Processes for Human Induced Pluripotent Stem Cell-Derived Spheroids

Human induced pluripotent stem (hiPS) cells have recently emerged as an important resource in the field of regenerative medicine. Along with recent successful clinical studies using hiPS cells, the realization of regenerative medicine using hiPS cells is in progress. Generally, hiPS cell-derived products are obtained via spheroids which are spherical cell aggregates.

In hiPS cell-derived product manufacturing, freezing processes are one of the most important steps because it is necessary for the transportation and preservation of the products. In general, process design of freezing for the spheroids has been explored based on trial-and-error experiments to find appropriate process conditions¹, although for future commercialization, there is a need to design freezing processes for hiPS cell-derived spheroids that can be operated stably and efficiently.

In the field of process systems engineering, several regenerative medicine-related processes have been investigated using model-based approaches, e.g., model-based assessment of temperature profiles in slow freezing for hiPS cells² and design space determination of mesenchymal stem cell cultivation processes³. However, model-based investigation of manufacturing processes related to hiPS cell-derived spheroids is still in infancy.

This work presents model-based assessment of design parameters in freezing processes for hiPS cell-derived spheroids. We first developed a physical model covering spheroid characteristics and freezing phenomena. To connect design parameters with cell quality, the developed model was extended to calculate the cell recovery ratio after thawing. The model parameters were estimated based on freezing experiments using hiPS cells. Given the three design parameters (the cooling rate, the spheroid radius, and the immersion time of the cryoprotective agent solution), the developed hybrid model can calculate the cell recovery ratio after thawing and the required freezing process time as quality and productivity objectives, respectively.

Model-based assessment of design parameters in the freezing process was demonstrated in a case study. As a result, the cooling rate and the spheroid radius have a significant impact on the cell recovery ratio after thawing. Based on the result, a design space of the freezing process was obtained, given a set of constraints such as the cell recovery ratio and the required freezing process time. The result would be useful in designing freezing processes for hiPS cell-derived spheroids. In the ongoing work, we are investigating bulk concentrations of the cryoprotective agent, and also developing a probabilistic design space by considering uncertainty.

References

1. Bissoyi A, Tomás RMF, Gao Y, Guo Q, Gibson MI. Cryopreservation of Liver-Cell Spheroids with Macromolecular Cryoprotectants. ACS Appl Mater Interfaces. 2023;15:2630–2638.

2. 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.

3. Hirono K, A. Udugama I, Hayashi Y, Kino-Oka M, Sugiyama H. A Dynamic and Probabilistic Design Space Determination Method for Mesenchymal Stem Cell Cultivation Processes. Ind Eng Chem Res. 2022;61(20):7009–7019.