Peripheral blood mononuclear cells (PBMCs) are a specific population of white blood cells circulating in the bloodstream. Defined by their unique morphology, they possess a single, round nucleus and include lymphocytes (T-cells and B-cells) and monocytes. As key components of the immune system, PBMCs serve as an accessible representation of a person’s immune health and response status. Isolating these cells from the complex mixture of whole blood is a foundational technique across immunology, infectious disease, and cancer research. This separation is necessary to study their functions, analyze their components, and prepare them for advanced therapeutic applications.
Why These Cells Matter
The accessibility of PBMCs makes them an unparalleled resource for monitoring and investigating the human immune system in both health and disease. Researchers use these isolated cells to track changes in immune cell populations, which can signal the progression of chronic illnesses such as HIV or various forms of cancer. Evaluating the immune response to new vaccines also relies heavily on PBMCs, as they contain the memory cells that indicate successful immunization.
The study of PBMCs provides direct insights into how the body is reacting to internal and external stimuli, allowing scientists to profile genetic and molecular shifts. This information is highly valuable in drug development, where PBMCs are used in assays to determine how an experimental compound might interact with the immune system. Furthermore, these cells are integral to the development of personalized cell therapies, including advanced treatments like Chimeric Antigen Receptor (CAR) T-cell therapy, where a patient’s own T-cells are genetically modified to fight cancer.
How Density Gradients Separate Cells
The separation of PBMCs from whole blood is achieved by exploiting a fundamental physical property: density. All components of blood, including red blood cells, granulocytes, and the mononuclear cells, have slightly different densities. A specialized separation medium, frequently a solution like Ficoll-Paque, is engineered to have a specific density, typically around $1.077 \text{ g/mL}$.
When a blood sample is layered over this medium and subjected to centrifugal force, the components travel downward until they reach a medium layer that matches their own density. Red blood cells and granulocytes, being denser than the separation solution, pass through the medium and pellet at the bottom of the tube. Conversely, the PBMCs, which have a lower density than the medium, accumulate as a distinct band at the interface of the plasma and the separation medium. The resulting stratification allows for the selective retrieval of the desired mononuclear cell population.
The Core Isolation Procedure
The isolation process begins with the preparation of the blood sample, which is typically collected with an anticoagulant to prevent clotting. Whole blood is first diluted, often at a one-to-one ratio with a balanced salt solution like phosphate-buffered saline (PBS), to decrease its viscosity and optimize the separation process. The diluted blood must then be precisely layered onto the dense separation medium inside a conical centrifuge tube.
Caution is necessary during this layering step to ensure the two liquids remain distinct and do not mix, which would compromise the density gradient. This is accomplished by gently pipetting the diluted blood down the side of the tilted tube, forming a clear boundary above the separation medium. Once layered, the tube is placed into a centrifuge and spun at a controlled speed, typically around 400 to 1,000 times the force of gravity ($g$), for a duration of 20 to 30 minutes at room temperature.
Crucially, the centrifuge brake is often turned off for the deceleration phase to allow the rotor to slow down gradually. Stopping the tube too abruptly could disturb the carefully formed layers, causing the separated components to remix and ruin the isolation. After centrifugation, a layer of clear plasma sits at the top, followed by a thin, cloudy white band of PBMCs—often called the buffy coat—at the interface with the separation medium.
The next step involves the careful harvesting of this PBMC layer using a pipette, avoiding the aspiration of the plasma above and the dense medium below. The collected cells are then transferred to a new tube and subjected to one or more washing steps with a buffer solution. These washes are performed by centrifuging the cells at a lower speed and removing the supernatant to eliminate residual plasma proteins, platelets, and any remaining gradient medium, ensuring a pure cell preparation for downstream applications.
Confirming the Cells Are Ready
After separation and washing, isolated PBMCs must undergo quality control to verify their suitability for research or clinical use. A primary check is cell viability, which determines the percentage of living cells in the final sample. This is commonly measured using the trypan blue exclusion method, where only dead cells with compromised membranes absorb the blue dye. A high-quality PBMC sample typically exhibits a viability of 90% or greater.
Researchers also perform a cell count to calculate the total yield of PBMCs obtained from the initial blood volume. This number helps standardize experiments and ensures consistency across different samples and isolation batches. Purity is assessed to confirm minimal contamination from unwanted cell types, such as red blood cells or the denser granulocytes that should have pelleted.
Once the cells pass these quality checks, they are either used immediately or prepared for long-term preservation. Cryopreservation is the standard storage method, involving resuspending the cells in a specialized freezing medium containing a cryoprotectant like dimethyl sulfoxide (DMSO). The cells are slowly frozen before transfer to liquid nitrogen for indefinite storage.