Optimization and evaluation of cryopreservation technology for lymphocytes

ObjectiveTo compare different cryopreservation methods for lymphocytes and explore an optimized preservation protocol. MethodsCryoprotectant 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. ResultsThe 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. ConclusionsBased 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.