The Changes Of Viability And Immunogenicity Of PBSC Collections After Long-Term Cryopreservation

Objective①To compare nucleated cell viability,CFU-GM-forming capability between the fresh and the long-term cryopreserved PBSC collections, and to study the factors which might correlate with the CFU-GM.②To compare immunophenotypes and functions of secrecting cytokines of lymphocytes in the collections between the fresh and frozen samples.③T o observe whether CIK cells could be prepared from long-term cryopreserved PBSC collections and still retain the cytotoxicity.Methods Among the total 47 samples of PBSC collections, frozen samples were divided into two groups: group 1( cryopreserved for 3~5 years ,n=26) and group 2(cryopreserved for 5~7 years, n=9); fresh samples (n=12)were as control. Flow cytometry method (FCM) based on 7AAD or caspase-3 staining was applied to assess the degree of cell necrosis(7AAD+) or apoptosis(caspase-3+) in cryopreserved and red-cell lytic collection samples. Lymphocyte immunophenotypes and the percentage of CD34 positive cells were also detected by FCM. CFU-GM culture was done to study the hematopoietic activity. The NC suspensions (5×105/ml) were stimulated by PHA (10μg/ml). And after simulated 48 h, the culture supernatants were collected to detect the concentrations of IL-2,interferon-γ(IFN–γ) and TNF-αby EILSA. Used 10 frozen samples and 7 fresh samples to expand CIK cells by IFN–γ, anti-CD3 monoclonal antibody and IL-2. On the 14th day, CIK cells were harvested to exam viability and analyse immunophenotypes and cytotoxicity.Results Both the necrosis and apoptosis levels of group1 and group2 were higher than control group(P<0.05); apoptosis cell of group2 was more than group1 (35.60%±17.51% to 24.14%±16.87%,P=0.031). CFU-GM/105NC decreased as the cryopreservation phase prolonged and it was the percentage of CD34+ cells, but not the cell viability correlated well with CFU-GM/105NC in the control and group1. There were no statistically differences in immunophenotypes and secreted cytokines among three groups. CIK cells from long-term frozen samples were expanded 41.8-fold(ranged from 11.50- to 80.64-fold),and 22.8-fold (ranged from 11.84- to 35.50-fold)from fresh samples. The percentage of CD8+ cell increased after cultured(P<0.05), but the purity of CD3+CD16/56+ cells didn't increase. The cytotoxicities of CIK cells against K562 and Raji cells weren't different between fresh and frozen samples.Conclusions Compared with the fresh PBSC collections, the long-term frozend ones have decreased NCs viabilities and have impairment of forming CFU-GM, which might relate to the decreased viability of CD34+ cells; but the lymphocyte immunophenotypes and the function of secreting cytokines: IL-2,IFN–γand TNF-αdon't differ. Cytotoxic CIK cells can be largely expanded from long-term frozen PBSC colltections.