Magnetic heating of nanoparticles as a scalable cryopreservation technology for human induced pluripotent stem cells

Abstract

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...

Chemical approaches to cryopreservation

“…Nanowarming exploits the local heating effect associated with magnetic nanoparticles in an alternating magnetic field, allowing homogeneous and rapid rewarming. Application of mesoporous silica-coated iron oxide nanoparticles have aided the cryopreservation of porcine arterial tissue (20 ml volume) 39 and magnetite (Fe 3 O 4 ) nanoparticles increased the cell viability of human induced pluripotent stem cells (hiPSCs) to 74.8% compared with 38.5% as assessed immediately post-thaw by Hoechst/SYTOX green staining 40 . Microporous silicon-coated iron oxide nanoparticles improved cryopreservation outcomes with samples as large as rat kidneys 41 .…”

Vitrification and Nanowarming of Kidneys

“…[13] More recently, Horie et al showed that human induced pluripotent stem cells (and cell aggregates) could be successfully vitrified and rewarmed with high viabilities in scaled up 20 mL CPA systems using RF nanowarming. [14] Using differential scanning calorimetry experiments, Xu et al showed that the presence of magnetic nanoparticles in CPA suppresses ice nucleation and growth during cooling and rewarming. [15] In parallel computational modeling efforts, Solanki et al and Eisenberg et al provide detailed analysis of the thermomechanical stresses during cooling and nanowarming in CPA solutions.…”

Investigation of Rapid Rewarming Chips for Cryopreservation by Joule Heating

“…Cryogenic vials are the most commonly used cryopreservation container, and their combination with water bath rewarming is currently considered the gold standard for the cryopreservation of most biological samples. However, cryogenic vials combined with convective rewarming can achieve a rewarming rate of only about ∼160 °C/min . This slow rewarming rate makes it possible for ice crystals to grow completely during the rewarming process, thus causing severe ice crystal damage to biological samples.…”