Peripheral Blood Mononuclear Cells, commonly known as PBMCs, are a group of specialized cells found circulating in the blood. These cells are characterized by having a single, round nucleus, distinguishing them from other blood components. PBMCs primarily include lymphocytes, such as T cells, B cells, and Natural Killer (NK) cells, along with monocytes, which are precursors to macrophages and dendritic cells. These cells collectively represent a significant portion of the body’s immune system, offering a valuable snapshot of an individual’s immune health and responses.
The Purpose of Isolating PBMCs
Separating PBMCs from whole blood allows scientists and medical professionals to study the human immune system. Isolating these cells helps researchers understand their behavior, function, and interactions in a controlled environment. This process is important for advancing immunology research and gaining insights into immune responses.
Isolated PBMCs are widely used to understand infectious diseases, including conditions like HIV and COVID-19, by analyzing how these cells respond to pathogens. They also contribute to the development of new vaccines, helping assess the effectiveness and immunogenicity of vaccine candidates. PBMCs play a role in personalized medicine, particularly in cancer immunotherapy, such as the development of CAR-T cell therapies. They are also used in drug development to evaluate the impact of potential treatments on immune cell function.
The Isolation Process
The most common laboratory method for separating PBMCs from whole blood is density gradient centrifugation, which leverages differences in cell densities. This technique works on the principle that when a sample is spun at high speed, components with different densities settle into distinct layers. A typical density gradient medium, such as Ficoll-Paque, is less dense than red blood cells and granulocytes but denser than PBMCs.
The isolation process begins by diluting a whole blood sample with a buffer like phosphate-buffered saline (PBS). This diluted blood is then carefully layered on top of the density gradient medium in a conical tube, ensuring the layers do not mix. The tube is then centrifuged, typically with the brake off, to prevent disturbance of the layers.
After centrifugation, distinct layers become visible. From top to bottom, these include a plasma layer, followed by a thin, whitish “buffy coat” containing the PBMCs at the interface of the plasma and the density medium. Below this are the clear density medium layer, and a pellet of red blood cells and granulocytes at the bottom. The buffy coat, containing the isolated PBMCs, is then carefully aspirated. To further purify the cells, collected PBMCs undergo washing steps to remove any remaining plasma, platelets, or density medium.
Verifying a Successful Isolation
After isolating PBMCs, confirming success involves two main quality control steps: determining cell yield and assessing viability. Cell counting quantifies the number of recovered cells from the initial blood sample. This is performed using a hemocytometer or automated cell counters.
Viability testing ensures isolated cells are alive and healthy for downstream applications. A common method is the trypan blue exclusion assay, where a blue dye is added to the cell suspension. Live cells exclude the dye, remaining unstained, while dead cells absorb it and appear blue. Red blood cell contamination can impact cell count accuracy and may require additional steps like red blood cell lysis.
Storing and Using Isolated Cells
Once PBMCs are isolated and their quality verified, they can be used immediately or prepared for long-term storage through cryopreservation. This process involves freezing cells in a protective medium to maintain viability and functionality. The medium contains a cryoprotectant like dimethyl sulfoxide (DMSO), which helps prevent ice crystal formation that damages cells during freezing.
For freezing, PBMCs are suspended in a cryopreservation medium, often containing DMSO and fetal bovine serum (FBS). Vials are placed into a controlled-rate freezing container, allowing the temperature to drop gradually. After an overnight stay in a -80°C freezer, cryovials are transferred to liquid nitrogen for long-term storage, ensuring cellular integrity.
Upon thawing, cryopreserved PBMCs can be utilized for various downstream applications. They are placed into cell culture to grow and proliferate. Researchers stimulate these cells with specific agents to study immune responses, such as cytokine production or differentiation patterns. Techniques like flow cytometry are also used to identify and quantify specific immune cell subtypes, providing detailed insights into cellular characteristics and functions.