Virtual Global Transplant Laboratory Standard Operating Procedures for Blood Collection, PBMC Isolation, and Storage

Abstract

Research on human immune responses frequently involves the use of peripheral blood mononuclear cells (PBMC) immediately, or at significantly delayed timepoints, after collection. This requires PBMC isolation from whole blood and cryopreservation for some applications. It is important to standardize protocols for blood collection, PBMC isolation, cryopreservation, and thawing that maximize survival and functionality of PBMC at the time of analysis. This resource includes detailed protocols describing blood collection tubes, isolation of PBMC using a density gradient, cryopreservation of PBMC, and thawing of cells as well as preparation for functional assays. For each protocol, we include important considerations, such as timing, storage temperatures, and freezing rate. In addition, we provide alternatives so that researchers can make informed decisions in determining the optimal protocol for their application.

FIGURE 1

Photograph of Ficoll layering. Image depicts a 50-mL conical tube after centrifugation for Ficoll layering. PBMC are in the white layer between the plasma and Ficoll.

FIGURE 2

Recovery of viable human PBMC frozen at different concentrations. PBMC from HVs and transplant recipients were isolated and frozen at the indicated concentrations by the University of Pennsylvania Human Immunology core. Viable cells were counted before freezing. Cells were frozen in a Mr. Frosty at −80°C and transferred to liquid nitrogen 1 to 2 days later. The number of viable cells post-thaw was divided by the number of viable cells prefreeze to determine % recovery (n = 41 samples thawed at 6 independent times). Patients were divided on the basis of (A) number of cells frozen and (B) source of sample. Differences in recovery were analyzed using an unpaired Student’s t test. For each comparison between groups, P was greater than 0.05. HV, healthy volunteers.

FIGURE 3

Comparison of cell viability of human PBMC cryopreserved with different devices. PBMC from healthy volunteers (n = 6, color-coded) were isolated and frozen using the indicated methods. Cell viability before freezing was measured for the baseline. Cells frozen in a controlled rate freezer were moved to a liquid nitrogen dewar upon completion of freezing cycle, whereas cells frozen in CoolCell and Mr. Frosty were transferred from −80°C to liquid nitrogen the following day. Ten days after transfer to liquid nitrogen, cells were thawed and viability measured. Results represent observations in at least 2 independent experiments.