Determining the effect of different storage temperatures on the live cell count of Peripheral Blood Mononuclear Cells (PBMCs)

Abstract

Cryopreservation of Peripheral Blood Mononuclear Cells (PBMCs) is an essential and common practice in conducting research. There are different reports in the literature attributing the need to preserve the integrity of the PBMCs. However, no specific suggestions have been made on the best preservation protocols to maintain both a high live cell count and integrity of the PBMCs.

Therefore, in this study the PBMCs were subjected to different storage temperatures that is; -20˚C, -80˚C and -20˚C followed by -80˚C for a maximum of 24hours each. Cell quantification was done by manual counting using an inverted microscope and trypan blue dye before and after storage to determine the percentage decrease in the live cell count of the PBMCs in order to confirm the effect of the different storage temperatures on the live cell count of the PBMCs. The percentage decrease in the live cell count of the PBMCs was computed and compared between the different storage temperatures. The effect of Red Blood Cells (RBCs) contamination on the live cell count of the PBMCs was also determined and this was done by isolating the PBMCs in two batches, with RBC lysis and without RBC lysis and then stored at the different PBMC storage temperatures.

The PBMCs that were stored at -20˚C had the greatest percentage decrease in live cell count while those stored at -80˚C had a moderate decrease in the percentage live cell count. However, the PBMCs that were stored at -20˚C followed by -80˚C had a slight percentage decrease in the live cell count.

There was no significant difference between the percentage decrease in live cell count of the PBMCs stored at -80˚C and -20˚C followed by -80˚C while a great significant difference was registered between the percentage decrease in live cell count of the PBMCs stored at -20˚C and -20˚C followed by -80˚C. Similarly, a high significant difference was also registered between the percentage decrease in live cell count of the PBMCs stored at -20˚C and -80˚C.

The PBMCs that were isolated without RBC lysis had a slightly higher percentage decrease in live cell count as compared to those that were isolated with RBC lysis. However, after storage there was no significant difference in the percentage decrease in live cell count of the PBMCs isolated with or without RBC lysis.

The percentage decrease in the live cell count of the PBMCs at the different storage temperatures was greatly attributed to cell apoptosis and cold shock that caused cell damage. RBC contamination had no effect on the live cell count of the PBMCs after storage because the RBCs may have undergone hemolysis due to the very low PBMC storage temperatures. The study thus showed that -20˚C followed by -80˚C is the best preservation protocol for the PBMCs and RBC contamination has a minimal influence on the live cell count of the PBMCs which is not significant.