Dual Suppression Effect of Magnetic Induction Heating and Microencapsulation on Ice Crystallization Enables Low-Cryoprotectant Vitrification of Stem Cell–Alginate Hydrogel Constructs

Stem cells microencapsulated in hydrogel as stem cell–hydrogel constructs have wide applications in the burgeoning cell-based medicine. Due to their short shelf life at ambient temperature, long-term storage or banking of the constructs is essential to the “off-the-shelf” ready availability needed for their widespread applications. As a high-efficiency, easy-to-operate, low-toxicity, and low-cost method for long-term storage of the constructs, low-cryoprotectant (CPA) vitrification has attracted tremendous attention recently. However, we found many cells in the stem cell–alginate constructs (∼500 μm in diameter) could not attach to the substrate post low-CPA vitrification with ∼2 M penetrating CPAs. To address this problem, we introduced nanowarming via magnetic induction heating (MIH) of Fe₃O₄ nanoparticles to minimize recrystallization and devitrification during the warming step of the low-CPA vitrification procedure. Our results indicate that high-quality stem cell–alginate hydrogel constructs with an intact microstructure, high immediate cell survival (>80%), and greatly improved attachment efficiency (by nearly three times, 68% versus 24%) of the encapsulated cells could be obtained post-cryopreservation with nanowarming. Moreover, the cells encapsulated in the cell–hydrogel constructs post-cryopreservation maintained normal proliferation under 3D culture and retained intact biological function of multilineage differentiation. This novel low-CPA vitrification approach for cell cryopreservation enabled by the combined use of alginate hydrogel microencapsulation and Fe₃O₄ nanoparticles-mediated nanowarming may be valuable in facilitating the widespread application of stem cells in the clinic.