Cryopreservation and long‐term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis effects on cell subset proportions and fluorescence intensity

Abstract

The effect of cryopreservation and long-term liquid nitrogen storage on peripheral blood mononuclear cell (PBMC) subsets was prospectively analyzed using monoclonal antibodies and flow cytometry. Brief cryopreservation did not significantly alter the proportion of positively stained cells for CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells. A small but statistically significant increase in the proportion of positive cells was observed for HLA-DR+ and HLe-1+ cells. Brief cryopreservation was associated with a decrease in the mean fluorescence intensity (MFI) values for CD3+, CD4+, and CD8+ cells; an increase in MFI values for CD14+ and HLA-DR+ cells; and no change for CD16+, CD19+, and HLe-1+ cells. There was no significant change in the proportion of CD3+, CD4+, or CD16+ cells during 20 months of storage in liquid nitrogen. Small but statistically significant decreases in the proportion of CD8+ and CD19+ cells were observed over the same interval, and the proportion of CD14+ cells (monocytes) was highly variable. Chronologic changes in fluorescence intensity during long-term storage were observed for all cell subsets except CD16+ and CD19+ cells. Cryopreservation is a valuable technique for long-term storage of viable cells. For many laboratory applications, the small changes noted in the present study will have no practical importance. However, for clinical and epidemiological investigations encompassing large numbers of samples, statistical techniques to adjust for small changes during storage should be considered.

Mesenchymal stem/stromal cell quality control: validation of mixed lymphocyte reaction assay using flow cytometry according to ICH Q2(R1)

“…First of all, we verified that the CTV-PBMC bank, which is critical in this method, was sufficiently stable over time and did not vary when changing the bank. We demonstrated its stability over 509 days, making it easy to use over a long time, consistent with the literature showing storage stabilities of PBMC cryopreserved in liquid nitrogen for several years [ 12 ]. Therefore, a CTV-PBMC bank can be used to qualify several productions of MSC batches in the context of one clinical trial.…”

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

“…However, our new method was demonstrated to detect ADCC activity more sensitively than the Cr 51 -assay, with individual ADCC activities being measureable even when frozen PBMCs were used. On the other hand, the phenotype of frozen PBMCs was reportedly changed 21 22 , or minimally altered within 2 months after being frozen 23 24 . Indeed, Mata et al suggested that PBMCs thawed and left overnight had high ADCC activity, though a change in the NK phenotype was observed after overnight incubation.…”

Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in 51Cr-release and CD107a assays

“…The cells were placed in a cryo 1°C freezing container (Thermo Fisher Scientific, Rochester, NY) in a −80°C freezer for 24 hr and then transferred into a liquid nitrogen freezer until use. The PBMC were used within 2 months of the date they were frozen to minimize phenotypic changes induced by the length of cryopreservation (Tollerud et al, 1991; Seale et al, 2008). Frozen PBMC were thawed in a 37°C water bath until a small amount of frozen material was left, then washed in complete media.…”