Why Separate Peripheral Blood Mononuclear Cells

Updated on Mar 25, 2024 By Jason Ellis, PhD Share

Within the human body, a sophisticated network of cells orchestrates the complex immune response against harmful pathogens and cancerous cells. Central to this defense mechanism is peripheral blood mononuclear cells (PBMCs), which include lymphocytes (T cells, B cells, NK cells) and monocytes. These cells are critical in mediating the body’s immune system and developing innovative treatments, including adoptive immunotherapies like chimeric antigen receptor (CAR) T cell therapy and T cell receptor (TCR) therapy.

The success of these therapies hinges on the efficient and gentle separation of pure and viable cells from a patient or donor’s blood. This multi-step process typically starts with separating PBMCs from other blood components like red blood cells (RBCs) and plasma. The PBMCs are then further sorted into different cell subtypes for use in research or cell therapy manufacture. Gentle cell separation is essential at every stage to preserve cell viability and function.

What Are Peripheral Blood Mononuclear Cells?

PBMCs are any cells found in the circulating blood that have a single round nucleus. By definition, this includes lymphocytes (T cells, B cells, natural killer cells, etc.) and monocytes (macrophages and monocyte-derived dendritic cells). It excludes platelets, red blood cells, and white blood cells with granular nuclei like neutrophils and eosinophils.

PBMCs circulate through arteries and veins that are easily accessible, such as in the arms and legs. Given their location, PBMCs often act as the body’s front line of defense against infection from cuts and other injuries. Researchers study PBMCs for regenerative therapy, diagnosing autoimmune diseases, and CAR T cell therapy.

PBMCs can be isolated from whole blood or via leukapheresis. Leukapheresis is a specialized medical procedure used to collect large volumes of white blood cells directly from a patient’s bloodstream, returning the rest of the blood components to circulation.

PBMC Isolation From Whole Blood

Without the capability of Leulopheresis, PBMCs can also be collected from whole blood using density gradient centrifugation, magnetic-activated cell sorting (MACS), or fluorescence-activated cell sorting (FACS).

Density Gradient Centrifugation

One of the most widely used methods for PBMC isolation is density gradient centrifugation. Using this technique, scientists add blood to a density medium in a tube and centrifuge it. The density medium is a pre-prepared sterile buffer solution with a very specific density optimized for layer stratification. Popular methods include using Ficoll-Paque medium or SepMate tubes.

The centrifugal force created at high spinning speeds separates the blood components based on their density compared to the solution. RBCs have a slightly higher density, spinning to the bottom of the tube, while PBMCs have a slightly lower density, leaving them to float on top of the density medium, and plasma will be in the top layer. After centrifugation, the layer containing PBMCs is carefully collected with a pipette for further use, leaving the unwanted RBCs and plasma behind.

Alternative Methods: FACS and MACS

While density gradient centrifugation is a cornerstone of PBMC isolation, recent advances have introduced more sophisticated methods, such as magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS uses magnetic beads coated with antibodies that specifically bind to certain cell types, allowing for the magnetic separation of targeted cells from the rest of the blood components.

FACS, on the other hand, uses flow cytometry. It involves tagging cells with fluorescently labeled antibodies and using automated laser technology to sort them based on their fluorescence intensity and light scattering properties. These methods offer higher specificity and purity of isolated PBMCs but come at the cost of increased complexity and potential cell stress due to the additional manipulation steps.

Advantages and Disadvantages of Current PBMC Isolation Techniques

Each PBMC isolation technique comes with its own set of advantages and disadvantages, impacting the choice of the method based on the specific requirements of the intended application.

• Density gradient centrifugation is simple and cost-effective, making it accessible to most laboratories. It is a robust method that can process large volumes of blood efficiently. However, it requires skilled personnel to perform accurately and can be time-consuming. Additionally, the physical stress from centrifugation may activate or damage some cells, potentially affecting their viability and functionality.
• Magnetic-activated cell sorting offers a higher degree of specificity and purity in the isolated PBMC population. MACS is particularly useful when isolating specific cell subsets, such as CD8+ T cells or NK cells. However, the need for specialized equipment and reagents increases the cost. Moreover, the binding of magnetic beads may interfere with cell surface receptors, potentially altering cell behavior or function.
• Fluorescence-activated cell sorting provides the highest specificity and allows for the automated sorting of cells into distinct populations based on multiple markers. This method enables detailed cellular studies and applications requiring high purity of certain cell types. Nevertheless, FACS is the most technically demanding and expensive option, requiring sophisticated equipment and expertise. The process is also slower and can induce significant stress on cells due to the use of lasers and droplet sorting, which might affect cell health.

Choosing the right PBMC isolation technique involves balancing the need for purity, specificity, and viability with the practical considerations of cost, time, and available resources. The potential for cell stress and damage inherent in some of these methods further highlights the need for gentle isolation techniques, especially for applications in adoptive cell therapy where cell integrity is paramount.

The Importance of Gentle Isolation

The method chosen for the isolation of PBMCs can significantly impact the viability and functionality of these cells, which in turn impacts their effectiveness in therapeutic applications and research. Gentle isolation methods are crucial for maintaining the integrity of PBMCs and minimizing cellular stress. This is especially important in the context of adoptive cell therapy, such as CAR T and TCR therapies, where the therapeutic efficacy depends on the cells’ ability to survive, proliferate, and function upon reinfusion into the patient.

By minimizing the manipulation and stress experienced by PBMCs during isolation, researchers and clinicians can obtain a healthier cell population with increased therapeutic efficacy.

Akadeum’s Microbubble Technology: A Gentle Solution

Akadeum Life Sciences offers an innovative alternative to traditional separation methods. Akadeum’s microbubble technology is gentle and effective. This technology allows microbubbles to bind and float undesired cells for easy removal, which provides “untouched” PBMCs with the least stress and the highest quality.

Our microbubble technology is:

• Gentle: The method is inherently gentle, reducing the risk of cellular damage and preserving the PBMCs’ native state. This is crucial for ensuring the cells retain their therapeutic potential and research relevance.
• Efficient: In addition to its gentleness, the technology is highly efficient at isolating subsets of PBMCs, providing high purity and high yield of targeted cell type This efficiency is key for both research study and therapeutic applications, where cell quantity and purity can be a limiting factor.
• Simple and scalable: Unlike some of the more equipment-intensive methods, Akadeum’s microbubble technology simplifies the isolation process, reducing the time for cell isolation to under 1hour. The process can be easily scaled up or down without the need for additional lab equipment, making the technology more easily integrated into your existing workflow.

By leveraging microbubble technology during PBMC isolation, Akadeum Life Sciences provides researchers and clinicians with a tool that not only enhances the quality of their isolated cell populations but also reduces the time and cost of cell separation. The technology’s gentle yet effective PBMC isolation opens new avenues for improving the outcomes of adoptive cell therapies.

Fast, Scalable, and Healthier Cells With Akadeum

By choosing Akadeum’s microbubble technology to optimize PBMC isolation, researchers and clinicians can ensure they are utilizing the most gentle and effective method available. We invite researchers developing adoptive cell therapies for cancer—such as CAR T therapy—and those involved in pre-clinical studies to explore how Akadeum’s microbubble technology can enhance their cell isolation processes. Adopting this technology means improving the quality of your PBMC preparations and contributing to the broader goal of advancing healthcare and scientific discovery.

For more information on how Akadeum’s technology can benefit your research or clinical applications—or to integrate our products into your cell separation workflow—contact Akadeum Life Sciences today. Let’s work together to push the boundaries of what’s possible in cell therapy and immunological research.