Figure 8.

Cryomicroscopic analysis and a schematic illustration of the possible mechanisms of enhancing cell vitrification by alginate hydrogel microencapsulation and nano-warming. A) Sample preparation for cryomicroscopic studies. A silicone film and coverslip are used to keep the thickness of experimental sample, including CPA, Fe₃O₄ NPs with CPA, and alginate hydrogel containing CPA. B) Typical images showing the freezing and warming processes of CPA solution, CPA solution with Fe₃O₄ NPs, and alginate hydrogel containing CPA. The red arrows indicate initial ice crystals. For the aforementioned three samples, the ice crystals appear at −70.8 °C, −61.9 °C and −84.1 °C, and continue to grow till −94.2 °C, −70.6 °C and −95.1 °C, respectively. Recrystallization occurs at −19.7 °C and −20.6 °C during warming CPA solution and CPA solution with NPs, respectively. Recrystallization does not occur during warming of alginate hydrogel containing CPA solution. C) Cells and cell-alginate hydrogel constructs at three different stages of the vitrification cryopreservation procedure: during cooling (a1-d1), during warming (a2-d2), and after warming (a3-d3). The alginate hydrogel microencapsulation may enhance vitrification during cooling and reduce devitrification/recrystallization during warming. The latter is further minimized by nano-warming via both the selective local and global heating effect of the Fe3O4 NPs under magnetic field. The nano-warming is crucial for cryopreservation of large cell-alginate hydrogel constructs by low-CPA vitrification.