Cryopreservation Of HUVECs With Alginate Hydrogel Microencapsulation And The Study Of Ice Suppression Mechanism

Cryopreservation is the primary method for achieving long-term storage of cells,tissues,and organs.Successful cryopreservation is critical for reproductive medicine,stem cell research,cord blood storage,and related biomedical research.However,during slow cooling and cryopreservation,the cells are susceptible to solute damage and ice crystal damage,while during vitrification preservation,the cells are susceptible to intracellular ice damage caused by devitrification.Therefore,it is very necessary to study the intracellular ice formation mechanism in the preservation process and propose a corresponding solution.In this paper,the probability of intracellular ice formation in PIECs with or without a protective agent was studied,and the parameters related to cell nucleation under different cooling rates were fitted,which reflected the effect of protective agent and cooling rate on intracellular ice formation.There are three different hypotheses for the formation mechanism of intracellular ice in the existing research.The most commonly used hypothesis is the catalytic nucleation hypothesis.Based on this hypothesis,the researchers have proposed the corresponding theoretical model of intracellular ice formation probability.It is based on the model.Secondly,an alginate hydrogel with a core-shell structure was prepared by electrostatic spray method for encapsulation and preservation of human umbilical vein endothelial cells.Existing studies have shown that alginate hydrogels can inhibit the appearance and growth of ice crystals during storage and can be used to encapsulate cells.Our experimental results show that core-shell microcapsules can achieve higher quality cell preservation under a lower concentration of protective agent(～3 M).At the same time,the low-temperature cold bench test of alginate capsules showed that the volume and boundary of the alginate capsules were not damaged during the f:reezing and rewarming process,and no formation of intracellular ice was observed in the encapsulated cells.It can be seen that the alginate hydrogel capsule has a good protective effect.Finally,the ice suppression mechanism of different concentrations of alginate solution and hydrogel was studied using a cryomicroscopic system.The experimental results show that the alginate solution can reduce the freezing point of the solution to a certain extent,and it will not change monotonously with the increase of concentration.Then,the effect of a certain thickness(300 μm)of alginate hydrogel on ice crystal formation was studied.The results show that the cross-linked hydrogel can greatly reduce the freezing point of the solution and slow down the growth rate and volume of the ice crystal.At the same time,the different crystallization mechanism of the protective agent and the alginate hydrogel was compared.This result once again verified that the alginate hydrogel has an anti-icing effect and can be applied to preserve other cells or tissues.