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 concentrations (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 volumetric concentrations 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.