Scale-up of production is needed for industrial applications and clinical translation of human induced pluripotent stem cells (hiPSCs). However, in cryopreservation of hiPSCs, successful rewarming of vitrified cells can only be achieved by convective warming of small volumes (generally 0.2 mL). Here, we present a scalable nano-warming technology for hiPSC cryopreservation employing inductive heating of magnetic nanoparticles under an alternating magnetic field. The conventional method by water bath heating at 37 °C resulted in a decrease of cell viability owing to devitrification caused by slow warming of samples with large volumes (≥ 20 mL). Nano-warming showed uniform and rapid rewarming of vitrified samples and improved viability of hiPSCs in the 20-mL system. In addition to single cells, hiPSC aggregates prepared using a bioreactor-based approach were successfully cryopreserved by the nano-warming technique. These results demonstrate that nano-warming is a promising methodology for cryopreservation in mass production of hipScs. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiP-SCs), are a promising cell source for regenerative medicine because of their unlimited proliferation potential and differentiation capability 1,2. The first patient was treated with an hESC-based cellular therapy product in a clinical trial for spinal cord injury in 2010 3. In 2014, the first patient received hiPSC-derived retinal pigment epithelial cells for macular degeneration 4. Although pluripotent stem cells have been generated at laboratory scale, large scale production processes by standardized and economically viable procedures and technologies will be required 5. One of the challenges in large scale expansion of pluripotent stem cells is suspension culture of cell aggregates in stirred bioreactors 6,7 in which single cells form cell aggregates in the presence of the small molecule Y27632 (Rho-associated coiled-coil kinase inhibitor) 8. Furthermore, bioreactor-based suspension culture of pluripotent stem cells can be used for large scale induction of functional cells such as iPSC-derived cardiomyocytes 9. In addition, a recent report suggests that cell aggregates can be used as building blocks for tissue engineering 10. One bottleneck in manufacturing pluripotent stem cells is robust cryopreservation, and large-scale cell cryopreservation will be mandatory for industrial applications and clinical translation. Cryopreservation is classified into two distinct methods: slow freezing and vitrification. In vitrification methods, cryoprotectant solutions with high cryoprotectant concentrations are used, and cells are rapidly frozen by direct immersion of the container in liquid nitrogen. Originally, vitrification methods were developed for cryopreservation of oocytes and embryos 11. Fujioka et al. developed DAP213, a cryoprotectant solution containing dimethyl sulfoxide (DMSO) for primate ESCs, and succeeded in cryopreservation of ESCs by vitrification of a 0.2-mL scale 12. H...

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Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.

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