Three-dimensional in vitro models constructed using cell spheroids or cell aggregates have gradually become more suitable in the field of tumor research. However

cryopreservation methods for cell spheroids are not ideal. The influence of the seeding density of A549 cells and cell culture days on the size and growth of A549 spheroids was studied. The results showed that when the seeding density was 2×104 cells /mL and the cells were cultured for three days

cell spheroids were in good condition and the microaggregates formed a compact structure with a diameter of 344.24 ± 0.74 μm

which is most suitable for cryopreservation. Then

the cooling box and program control cooling instrument were used to cryopreserve the A549 spheroids. After rewarming

the vitality and proliferation rate of cell spheroids were much lower than those of fresh cell spheroids. Plastic straws and glass capillaries were used to quickly freeze or vitrify the A549 spheroids. When glass capillaries were used as the freezing carrier and 20% EG + 20% Me2SO + 0.5 mol/L trehalose was used as the cryoprotectant

the relative vitality of A549 spheroids to fresh spheroids was 94.59% ± 9.23%

and the three-day culture proliferation rate was closest to that of the fresh group. Finally

the cooling rate of the freezing carrier and the critical cooling rate of the cryoprotectant were measured

and the thermodynamic mechanism of cell spheroid cryopreservation was analyzed.