PBMC Processing for Human Challenge Studies
When conducting Human Challenge Studies, WCCT processes peripheral mononuclear blood cells (PBMCs) for future testing. PBMC processing is a unique capability and one of WCCT’s core competencies. Through effective harvesting, processing, and analysis of PBMCs, researchers are able to evaluate immune responses to therapeutic agents and gain a deeper understanding of immune system function pre-dose, post-dose, and at other integral stages of testing.
There are three methods of PBMC isolation:
- Cell Preparation Tube (CPT)
- Lymphoprep Tube
WCCT primarily uses the CPT Method for isolating PBMCs during human challenge studies. The process for this is as follows:
1. Blood is collected into an 8.5 mL CPT tube.
2. These tubes come laced with a variety of anticoagulants including Heparin, Lithium, and Sodium Citrate.
3. CPT tube is then spun down in the centrifuge to separate red blood cells and granulocytes from PBMCs. Following centrifugation, serum and PBMCs remain above the thick resin layer which prevents any mixing with other blood components.
4. Once separated, supernatant is removed and remaining PBMC cells are transferred to a 50mL conical tube and re-suspended in a volume of PBS (Phosphate Buffered Saline).
5. Sample is Inverted and spun down again to form a cell pellet.
6. PBS is decanted and an equal volume of PBS is added. The cell pellet is broken up and re-suspended in PBS.
7. Sample is spun down once more as a final wash.
8. Once Again, PBS is removed and PBMC pellet is broken up and re-suspended in a measure of fetal bovine serum. (FBS is used to re-suspend PBMC cells because it provides an environment similar to human serum.)
9. A freezing media is then prepared using a measure of FBS and DMSO so that the concentration is equal to 20% DMSO.
10. Several cryovials are prefilled with this mixture and an equal measure of re-suspended PBMC resuspension is added bringing the concentration of DMSO to 10%.
11. Samples are capped and immediately transferred to a Mr. Frosty and placed in -70 storage. ( The purpose of using the Mr. Frosty is to allow the samples to cool at a rate of -1 Degree Celsius per minute. A gradual freezing process is necessary to maintain cell viability.)
In the wake of collaborations with the Stanford School of Medicine and various industry partners, WCCT Global has recognized the need to routinely isolate Peripheral Blood Mononuclear Cell (PBMCs) to characterize the human immune response to disease and various immune-targeted therapeutic agents.