Effects of Temperature, Length of Frozen Storage, and the Freezing Container on the Quality of Human Peripheral Blood Mononuclear Cells

Mononuclear cells were isolated from ficoll-hypaque treated cellular residue obtained during plateletpheresis. The peripheral blood stem cells were frozen and stored in provials or in plastic bags for as long as 2.4 years. We studied the effects of the temperature and length of frozen storage and the freezing container on the in vitro recovery and membrane integrity of the mononuclear cells in CFU-GEMM tissue culture, The peripheral blood mononuclear cells PBMC from each healthy donor were resuspended in autologous ACD plasma, and then treated with 27 DMSO in saline to achieve a final DMSO concentration of 10. The mononuclear cells were divided among a polyvinylchloride plastic bag, a polyolefin plastic bag, and 4 polyethylene provials. The cells in the polyvinyl chloride plastic bag were frozen at -80 C and the cells in the polyolefin plastic bag were frozen at -135 C the cells in the provials were frozen at -80 C, -135 C, -150 C, or -197 C. Mechanical refrigeration was used for -80 C and -135 C storage the vapor phase of liquid nitrogen was used for -150 C storage and the liquid phase of liquid nitrogen was used for -197 C storage. The mononuclear cells were frozen and stored for 1.5 years or 2.4 years, thawed, washed, and tested. The samples frozen in plastic bags in mechanical refrigerators at -80 C and -135 C for 2.4 years exhibited excellent in vitro recovery and viability values of greater than 90. Mononuclear cells frozen and stored in plastic bags exhibited no loss of CFU-GEMM activity after 1.5 years at -80 C or -135 C 40 loss after 2.4 years of -80 C storage and maintenance of CFU-GEMM activity after 2.4 years of storage at -135 C. After frozen storage at -80 C, -135 C, -150 C or -197 C for 1.5 or 2.4 years, the PBMC in provials exhibited significantly lower in vitro recovery values than the PBMC in plastic bags, although viability values were not significantly lower.