Objective To compare different cryopreservation methods for lymphocytes and explore an optimized preservation protocol.

Methods Cryoprotectant formulations containing different concentrations of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), polyethylene glycol (PEG), and bovine serum albumin (BSA) were screened for lymphocytes to identify two optimized formulations: experimental group 1 (C11): (7.5% DMSO + 10% FBS + 2.5% PEG + 2% BSA) and experimental group 2 (C21): (7.5% DMSO + 2.5% PEG + 2% BSA). On this basis, cryopreservation performance was compared between the two optimized cryoprotectant formulations paired with their respective optimal cooling rates (C11: −3 °C/min; C21: −1 °C/min) and the conventional cryopreservation method control group (C0): (10% DMSO + 90% FBS) with gradient cooling. After 5 days of cryopreservation, cells were thawed to assess preservation quality indicators in terms of cell viability and functionality across groups.

Results The cell recovery rate at 0 h post-thawing in the control group (C0) (65.70 ± 1.06)% was lower than that in the experimental group 1 (C11) (82.45 ± 3.27)%, t = −8.44, P < 0.05 and experimental group 2 (C21) (83.96 ± 0.81)%, t = −23.72, P < 0.05. The apoptosis rate at 0 h post-thawing in the control group (C0) (6.93 ± 0.55)% was higher than that in the experimental group 1 (C11) (5.30% ± 0.56)%, t = 3.61, P < 0.05 and experimental group 2 (C21) (4.93 ± 0.45)%, t = 4.87, P < 0.05. Moreover, the differences between groups were statistically significant.

Conclusions Based on multifactorial screening and optimization, the experimental group 2 using a serum-free cryoprotectant with a cooling rate of −1 °C/min outperforms the conventional cryopreservation method in terms of cell quality. This preservation protocol provides a scientific reference for the cryopreservation of lymphocytes.