Peripheral blood mononuclear cells (PBMCs) are an important immune cell resource. However

traditional slow-freezing methods delay PBMCs proliferation and cause damage to T cell subsets. Therefore

there is a clear need to develop an alternative cooling procedure that can effectively preserve the viability and function of PBMCs. In this study

we optimized the cryopreservation process of PBMCs using ultrasonic ice seeding and analyzed T cell subtypes post-cryopreservation through flow cytometry. An ultrasonic ice-seeding apparatus was constructed to achieve contactless ice seeding through the combination of an ultrasonic generating device and a controlled rate freezer. The results showed that the cooling procedure involving ultrasonic ice seeding exhibited superior efficacy compared to the conventional slow freezing approach. Following cryopreservation

the viability and cumulative proliferation of PBMCs were 94.97% and 204.47%

respectively. The proportion of naive T cells (Tn) after cryopreservation and thawing accounted for up to 18.35%. By incorporating ultrasonic ice seeding

the optimized cryopreservation procedure enhanced the post-thaw viability

cumulative proliferative capacity

and proportion of T cell subtypes in PBMCs

providing a novel and effective approach for PBMCs cryopreservation.