Why PBMCs are Gaining Popularity?
Peripheral blood mononuclear cells (PBMCs) are immune response cells that include lymphocytes, monocytes and dendritic cells. PBMCs are widely used in clinical research because they provide a convenient, minimally invasive way to study the immune system and its response to diseases, infections and therapies.
Here are the Key Reasons for their Popularity
Relevance to Immunity and Disease
They play a critical role in immune responses, making them valuable to research into autoimmune diseases, cancer immunotheraphy, infectious diseases, and vaccine responses.
Diverse Cell Types
PBMCs include immune cells like T cells, B cells, NK cells, and monocytes; offering insights into multiple aspects of immune function in a single sample.
In Vitro Modeling
PBMCs can be used for in vitro experiments to test drug responses, study cell behavior, and observe cellular interactions in a controlled setting.
Standardization and Consistency
Techniques for isolating, storing and analyzing PBMCs are well-established, enabling reproducible results and facilitating comparisons across studies.
Accessibility
PBMCs can be isolated easily from blood samples, making it simpler to obtain them repeatedly for monitoring changes over time.
Why Use PBMCs?
PBMCs are useful in understanding various physiological and pathological mechanisms in humans and animals. The following are applications of PMBCs and reasons why animal PBMCs have become widely used in recent years:
Immune Response Modelling
Representing different immune cell types, PBMCs are integral in the study of immune responses such as cytokine release and cellular interactions.
Drug Screening and Immunotherapy Testing
They can be used to test new drug efficacy and toxicity before proceeding with whole-animal studies.
In Vitro Analysis
Isolation of PBMCs from blood samples allows creation of in vitro cultures for investigating immune cell functions such as differentiation, proliferation and cytokine production while eliminating the need for invasive procedures that harm animals.
Genetic Studies and Biomarker Discovery
PMBCs from different animal species helps in identifying conserved immune responses that demonstrate immune function across various animals.
Translatability to Human Studies
Human and animal PMBCs share characteristics that enable extrapolation of either studies such that preclinical studies can predict the performance of treatments in human subjects.
Reduced Animal Use
In vitro studies using PBMCs can reduce the number of animals needed in experiments.
Disease Modeling
They can be used to test new drug efficacy and toxicity before proceeding with whole-animal studies.
PBMC Preparation
Animal Blood Collection
As required by institutional and ethical guidelines, the animal is anesthetized before blood collection. Blood collection can be accomplished depending on the size of the animal. For small animals such as rats and mice, blood is typically collected from the tail vein, retro-orbital sinus, or by cardiac puncture. For larger animals such as non-human primates, blood is commonly collected from a vein, mainly the saphenous or femoral vein. To prevent clotting, an anticoagulant is added immediately to the blood. Heparin or EDTA is usually used as anticoagulant. The blood is then gently mixed by inverting the tube several times. Vigorous shaking is avoided to prevent hemolysis.
Dilution of Blood
Density Gradient CentrifugationAfter addition of the coagulant, the blood is diluted with an equal volume of PBS or sterile saline. This step enables the reduction of cell density to facilitate better separation in the gradient. The solution is then gently mixed by inverting the tube.
Density Gradient Centrifugation
A density gradient medium is prepared by adding Ficoll-Paque or Histopaque to a sterile 15 mL or 50 mL centrifuge tube. Approximately 3 mL of density gradient medium per 10 mL of diluted blood is then used. The diluted blood is then layered carefully on top of the density gradient medium. By adding blood along the side of the tube using a pipette, mixing of the mixture is avoided. Next, the mixture is centrifuged at 400 x g for 20-30 minutes at room temperature. Gentle layer separation is ensured by not using the centrifuge brake.
Collection of PBMC Layer
The PBMC layer is identified based on its position alongside three other components. The top layer is composed of plasma. The second layer is thin and white, representing the PBMC layer. The third layer is made up of the density gradient medium which is either Ficoll or Histopaque. Finally, the fourth and bottom layer is composed of red blood cells. The second layer, which is the PBMC layer, is carefully harvested using a pipette and then transferred into a new, clean 15 mL centrifuged tube.
Washing the PBMCs
Once the PBMCs have been collected, PBS or culture medium is added to dilute the Ficoll or Histopaque. Next, the solution is centrifuged at 300 x g for 10 minutes at room temperature to pellet the PBMCs. After discarding the supernatant, the cell pellet is gently resuspended in fresh PBS or culture medium. This is followed by two more steps of washing to make sure no Ficoll/Histopaque and other contaminants remain.
Counting and Viability Assessment
To measure viability, cells are first counted using a hemocytometer or an automated cell counter. The cell concentration can be adjusted depending on downstream experiment requirements. Then, cell viability assessment is performed using tryphan blue staining or another similar method. Ideally, the viability of PBMCs for functional assays should be above 90%.
Storage/Immediate Use
If the freshly collected PBMCs are to be used immediately, cells are resuspended in an appropriate medium or buffer for further processing for techniques such as flow cytometry, cell culture, etc. If the PBMCs are to be stored for later use, the cells are frozen in a cryoprotective medium such as 10% DMSO in fetal bovine serum and then gradually cooled to -80°C before transferring to liquid nitrogen for long-term storage. We cryopreserve the cells in serum-free cryopreservation media to prevent potential effects of growth factors before and during international shipping. Let us know if you have special requests and we will be glad to accommodate them.
PBMC-Based Assays
PBMCs are a versatile sample type in preclinical research due to their role in the immune system. Assays using PBMCs help assess immune function, response to therapies, and disease pathophysiology.
Animal PBMCs are also commonly used in preclinical Safety, Toxicology and Translational research to help select the right in vivo model for late preclinial studies.
The following are common PBMC-based assays in preclinical research.
Flow Cytometry
This assay quantifies and analyzes various immune cell subsets within PBMCs, such as T cells, B cells, NK cells, and monocytes. Flow cytometry is widely used to determine immune status in diseases like HIV or cancer, evaluate immune responses to therapies, and track cell phenotypes in clinical trials.
ELISPOT (Enzyme-Linked Immunospot) Assay
ELISPOT measures the frequency of cytokine-secreting cells, indicating immune activation. It is often used in vaccine trials or infectious disease research to assess cellular immune responses by quantifying cytokines like IFN-γ, which indicates T-cell activation.
Intracellular Cytokine Staining (ICS)
ICS is used to detect cytokine production within individual cells using flow cytometry. This assay helps identify specific functional responses, such as Th1, Th2, or Th17 responses, by measuring cytokines like IL-2, IFN-γ, and TNF. It’s particularly valuable in vaccine and immunotherapy studies.
Proliferation Assays
These assays measure the ability of PBMCs to proliferate in response to specific antigens or mitogens. They are used in immunological research to assess immune responsiveness in autoimmune diseases, vaccine trials, or immunodeficiencies. Proliferation assays help determine immune system activation and potential deficiencies in cell-mediated immunity.
Cytotoxicity Assays
These assays assess the cytolytic activity of PBMCs, particularly NK cells and cytotoxic T cells, against target cells. They are instrumental in cancer and viral research for evaluating how well immune cells can kill infected or tumor cells.
Gene Expression Profiling
The purpose of this assay measures gene expression changes in PBMCs using techniques like qPCR or RNA sequencing. Applications in autoimmune and infectious disease research include revealing insights into immune response mechanisms as well as helping identify biomarkers for disease progression and treatment response.
Single-Cell RNA Sequencing (scRNA-seq)
This technique analyzes gene expression at the single-cell level to characterize the transcriptome of individual cells. Its applications in immunology and oncology include exploration of the heterogeneity of immune cell populations, particularly in response to treatments or in different disease states.
Mixed Lymphocyte Reaction (MLR)
MLR is used to measure the response of immune cells to foreign cells. Its typical application is in transplant immunology which is to assess compatibility, by helping understand immune responses to allogeneic tissues and immune tolerance.
T-Cell Receptor (TCR) Sequencing
TCR is a technique for identifying the diversity and clonality of T-cell receptors in PBMCs.
It is valuable in cancer immunotherapy research for tracking immune responses and to understand T-cell repertoire dynamics in response to treatments, such as checkpoint inhibitors.
These PBMC assays offer a comprehensive set of tools for understanding immune function and disease mechanisms of humans and animals in clinical research and are invaluable for monitoring immune responses to therapies across various fields.
Have you decided which PBMCs to use and what assays they are for?
Get High-Quality PBMC Samples
High-quality samples not only ensure reliable and reproducible results in research and clinical applications, they also come with the following advantages:
Superior Cell Viability and Function
High-quality samples mean cells stay alive and functional for the duration of the study, which is necessary for achieving accuracy of parameters involving proliferation, cytokine production, immune activation and other cellular responses.
Reproducibility of Results
Consistent results across multiple time points are very important when tracking immune responses or disease progression.
Increased Sensitivity in Biomarkers Detection
Quality PBMC samples help ensure that biomarkers, such as cytokines, are detected even at low levels to better assess disease behavior and treatment responses.
Enhanced Response to Stimuli
High-quality samples tend to respond more accurately to functional assay stimuli such as antigens or drugs thus providing a more precise picture of immune activation, drug efficacy, or toxicity.
Efficient Use of Resources
Excellent samples lower the odds of failing assays and losing data, thus saving time, effort, and money.
Minimizes Sample Replication
High-quality cells can be advantageous when dealing with limited sample volumes such as in rare human specimens and small animals.
Reduces Contamination or Artifacts
Superior samples are less likely to have contamination or impurities such as dead cells or debris that could interfere with analyses and muddle results.
Better Cryopreservation Potential
High-quality PBMC samples perform well during cryopreservation as well as later use since high initial viability cells can survive freezing and thawing processes better.
We Ensure Quality
To see to it that the minimum cell number post-thaw is achieved for each unit shipped, we overfill containers with cells by 50%.
We cryopreserve the cells in serum-free cryopreservation media to prevent potential effects of growth factors before and during international shipping via our logistics partners.
We typically ship cells that are in stock within two days. New projects can be delivered within 2 weeks for minipig/pig/beagle/monkey/llama and 3 weeks for alpaca PBMCs.
We ship cells Europe-wide within 24-48 hours, and can ship intercontinentally with dry shipper.
Still undecided which species to use?
References
Fuss, I. J., Kanof, M. E., Smith, P. D., & Zola, H. (2009). Isolation of whole mononuclear cells from peripheral blood and cord blood. Current protocols in immunology, Chapter 7, 7.1.1–7.1.8. https://doi.org/10.1002/0471142735.im0701s85
Lyu, M., Shi, X., Liu, Y., Zhao, H., Yuan, Y., Xie, R., Gu, Y., Dong, Y., & Wang, M. (2023). Single-cell transcriptome analysis of H5N1-HA-stimulated alpaca PBMCs. Biomolecules, 13(60). https://doi.org/10.3390/biom13010060
Riedhammer, C., Halbritter, D., & Weissert, R. (2016). Peripheral Blood Mononuclear Cells: Isolation, Freezing, Thawing, and Culture. Methods in molecular biology (Clifton, N.J.), 1304, 53–61. https://doi.org/10.1007/7651_2014_99
Yu, S., Xiong, G., Zhao, S., Tang, Y., Tang, H., Wang, K., Liu, H., Lan, K., Bi, X., & Duan, S. (2021). Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review). International journal of molecular medicine, 47(2), 444–454. https://doi.org/10.3892/ijmm.2020.4817
