Diverting the food-freezing technology improves the cryopreservation efficiency of induced pluripotent stem cells and derived neurospheres.

Introduction: Recent advances in induced pluripotent stem (iPS) technology and regenerative medicine require effective cryopreservation of iPSC-derived differentiated cells and three-dimensional cell aggregates (eg. Spheroids and organoids). Moreover, innovative freezing technologies for keeping food fresh over the long-term rapidly developed in the food industry. Therefore, we examined whether one of such freezing technologies, called "Dynamic Effect Powerful Antioxidation Keeping (DEPAK)," could be effective for the cryopreservation of biological materials. Methods: We evaluated the efficiency of cryopreservation using DEPAK and Proton freezers, both of which are used in the food industry, compared with conventional slow-freezing methods using a programmable freezer and a cell-freezing vessel. As they are highly susceptible cells to freeze-thaw damage, we selected two suspension cell lines (KHYG-1 derived from human natural killer cell leukemia and THP-1 derived from human acute monocyte leukemia) and two adherent cell lines (OVMANA derived from human ovarian tumors and HuH-7 derived from human hepatocarcinoma). We used two human iPS cell lines, 201B7-Ff and 1231A3, which were either undifferentiated or differentiated into neurospheres. After freezing using the above methods, the frozen cells and neurospheres were immediately transferred to liquid nitrogen. After thawing, we assessed the cryopreservation efficiency of cell viability, proliferation, neurosphere formation, and neurite outgrowth after thawing. Results: Among the four cryopreservation methods, DEPAK freezing resulted in the highest cell proliferation in suspension and adherent cell lines. Similar results were obtained for the cryopreservation of undifferentiated human iPS cells. In addition, we demonstrated that the DEPAK freezing method sustained the neurosphere formation capacity of differentiated iPS cells to the same extent as unfrozen controls. In addition, we observed that DEPAK-frozen neurospheres exhibited higher viability after thawing and underwent neural differentiation more efficiently than slow-freezing methods. Conclusions: Our results suggest that diversifying food-freezing technologies can overcome the difficulties associated with the cryopreservation of various biological materials, including three-dimensional cell aggregates.