Description
Human induced pluripotent stem cells (hiPSCs) are an attractive cell source for
regenerative medicine. For its widespread use as a starting material, a robust
storage and distribution system in the frozen state is necessary. For this system,
managing transient warming during storage and transport is essential, but how
transient warming affects cells and the mechanisms involved are not yet fully
understood. This study examined the in uence of temperature cyclings
(from−80°C to−150°C) on cryopreserved hiPSCs using a custom-made cryo
Raman microscope, ow cytometry, and performance indices to assess viability.
Raman spectroscopy indicated the disappearance of mitochondrial cytochrome
signals after thawing. A reduction in the mitochondrial membrane potential was
detected using ow cytometry. The performance indices indicated a decrease in
attachment ef ciency with an increase in the number of temperature cycles. This
decrease was observed in the temperature cycle range above the glass transition
temperature of the cryoprotectant. Raman observations captured an increase in the
signal intensity of intracellular dimethyl sulfoxide (DMSO) during temperature cycles.
Based on these results, we proposed a schematic illustration for cellular responses to
temperature uctuations, suggesting that temperature uctuations above the glass-
transition temperature trigger the movement of DMSO, leading to cytochrome c
oxidation, mitochondrial damage, and caspase-mediated cell death. This enhances
our understanding of the key events during cryopreservation and informs the
development of quality control strategies for hiPSC storage and transport.