(410g) Slow Freezing Process Design for Human Induced Pluripotent Stem Cells By Modeling Intracontainer Variation

Authors: 
Sugiyama, H., The University of Tokyo
Hayashi, Y., The University of Tokyo
Horiguchi, I., Osaka University
Kino-oka, M., Osaka University
Human induced pluripotent stem (hiPS) cells are the emerging source of regenerative medicine products. Along with the successful clinical studies for treating diseases such as Parkinson’s disease, or spinal cord injuries, the demand of hiPS cells is increasing. This trend indicates the necessity for establishing cryopreservation processes of hiPS cells for storage and transportation. Generally, there are two methods for freezing cells: slow freezing and vitrification. For hiPS cells, it is planed that the liquid containing hiPS cells are filled in to a container such as vial, cryopreserved, and then thawed at the point of use. Here, slow freezing, as compared to vitrification, can benefit from scalability, simplicity in operation, and no direct contact with freezing agent, such as liquid nitrogen. However, the process could suffer from the quality variation inside the vial (i.e., intracontainer), between vials (i.e., intercontainer), and between and among freezers (i.e., intra- and interfreezer).

We present a model-based design of slow freezing processes for hiPS cells considering the intracontainer quality variation. A single-cell model was developed by integrating models describing a radial and temporal temperature profile, cell volume change through transmembrane water transport, and intracellular ice formation during slow freezing. Given the cooling rate of the freezer, the vial diameter and material, and the cryoprotective agent type, the model can produce the maxima of cell volume change and the intracellular ice crystal volume as the cell quality indicators, and the required freezing time as the productivity indicator.

The developed model showed sufficient performance compared with a recently reported experimental study on hiPS cells. Upon this confirmation, the model was applied for three design cases. The choice of a cryoprotective agent, cooling rate, and vial diameter affected the quality evaluation more than the choice of the vial material. When considering the productivity as the design objective, the optimal vial diameter changed depending on the constraints of cell demand and the acceptable intracellular ice crystal volume. In the ongoing work, we are performing rigorous performance assessment of the model by extensive experiments. Advanced process design and optimization, e.g., dynamic temperature optimization, integrated design of freeze/thaw process, is another ongoing subject.