Examining Ionic Cross-linked Alginate Hydrogels in Cell Culture and Cryopreservation

In recent years, hydrogels have exhibited considerable advantages in cell and organoids culture as well as cryopreservation, particularly in cell cryopreservation. In this study, the spray method was selected, and ferric iron and calcium were used as cross-linking agents. The hydrogels were prepared using a low-cost and readily available airbrush. Subsequently, calcium alginate and iron alginate hydrogels were prepared, and HEK293T cells and HepG2 cells were encapsulated using this method. The study also investigated the impact of cross-linking solutions on cell viability during encapsulation, revealing that longer crosslinking times led to greater cell damage. Additionally, it was observed that crosslinking with sodium alginate solution reduced cell damage in the crosslinking solution. Furthermore, hydrogels crosslinked with CaCl₂ (0.2 mol/L) using sodium alginate solution at various mass fraction (1%, 1.5%, and 3%) were prepared. HEK293T cells were encapsulated and cultured for 7 d under these conditions, and successful cell culture was observed across all concentrations. Finally, the viability of cell cryopreservation after encapsulation was examined. The results showed that the viability of HEK293T and HepG2 cells, under the precondition of using two different volume fraction of dimethyl sulfoxide as protective agents, was significantly higher than that of the unencapsulated group. Specifically, for HEK293T cells before encapsulation, cell viability was 33.16% ± 2.70% (10% DMSO) and 16.75% ± 2.3% (5% DMSO). After encapsulation, cell viability increased to 76.51% ± 5.32% (10% DMSO) and 60.86% ± 2.41% (5% DMSO). Similarly, for HepG2 cells, cell viability increased from 48.93% ± 3.06% (10% DMSO) and 36.22% ± 2.54% (5% DMSO) to 78.79% ± 4.43% (10% DMSO) and 64.64% ± 3.13% (5% DMSO) after encapsulation. Moreover, when using ethylene glycol (1 mol/L) + propylene glycol (1.5 mol/L) + trehalose (1 mol/L) as a protective agent, the viability of HEK293T cells after vitrification increased from 33.5% ± 0.8% to 79% ± 3.76% after encapsulation. These results strongly indicate that encapsulated cells exhibit significantly higher viability after cryopreservation compared to unencapsulated cells, emphasizing the protective capacity of ionic cross-linked alginate hydrogels during cryopreservation.