Why Many Choose Cynomolgus PBMCs
Cynomolgus monkeys (Macaca fascicularis), also known as long-tailed or crab-eating macaques, are the most widely used nonhuman primate specimens for biomedical research. The use of cynomolgus macaques in research began in the mid-20th century as the demand for non-human primates grew in fields like infectious disease, pharmacology, and toxicology. Their availability in the wild, especially across Southeast Asia, and their manageable size made them a practical choice compared to larger primates like rhesus macaques or chimpanzees.
Cynomolgus PBMCs have an established role in preclinical research due to Cynomolgus’ genetic and immunological resemblance to humans. The following are some key benefits of using cynomolgus PBMCs in clinical research.
Similarity to Humans
Cynomolgus monkeys and humans share a very high degree of genetic similarity, especially in the components of their immune systems, Cynomolgus PBMCs are known to be highly relevant for investigating human immune diseases and responses. This makes Cynomolgus results more predictive of human outcomes compared to results derived from rodent models.
Ideal for Immunological Studies
PBMCs from cynomolgus monkeys include immune cells such as T cells, B cells, NK cells, and monocytes, which are vital for investigating autoimmune diseases, immune responses, and immune cell interactions. This makes them a suitable model for understanding the immunogenicity and immunotoxicity of drugs.
Predictive Power in Drug Development
Cynomolgus PBMCs allow researchers to assess the efficacy and safety of immunotherapies, biologics, and other drugs targeting the immune system before moving into human clinical trials.
High Relevance for Monoclonal Antibody Studies
Cynomolgus monkeys have receptors and immune targets resembling humans, making their PBMCs widely accepted for assessing the efficacy, pharmacodynamics, and safety of monoclonal antibodies and other biologic therapies.
Zoonotic Disease Research
– Cynomolgus PBMCs are highly valuable for studying zoonotic diseases such as tuberculosis and simian immunodeficiency virus (SIV), which is comparable to HIV in humans. These studies help in the development of vaccines and treatments for infectious diseases with human relevance.
Regulatory Acceptance
Regulatory agencies, such as the European Medicines Authority, recognize cynomolgus monkey studies as relevant for assessing the immunotoxicity and safety of new drugs and biologics. This speeds up the approval process by providing data from a model that regulators consider predictive of human responses.
Published Research Using Cynomolgus PBMCs
The study by Qiu et al. (2009), titled “Mucosal Immunization of Cynomolgus Macaques with the VSVΔG/ZEBOVGP Vaccine Stimulates Strong Ebola GP-Specific Immune Responses” investigates the efficacy of a live attenuated recombinant Vesicular Stomatitis Virus (VSV)-based vaccine against Zaire Ebola virus (ZEBOV) in cynomolgus macaques. The vaccine, VSVΔG/ZEBOVGP, expresses the glycoprotein of ZEBOV and was tested using three delivery routes: intranasal (IN), oral (OR), and intramuscular (IM).
Key Highlights of Cynomolgus PBMCs
Protection from Lethal Challenge
- All vaccinated macaques were fully protected from a high-dose ZEBOV challenge, showing no clinical signs of disease, while control animals succumbed to infection.
Immune Response
- The vaccine elicited strong GP-specific humoral and cellular immune responses across all routes of administration.
- Long-term memory B and T cell responses were observed, indicative of robust immunity post-vaccination.
Comparison of Delivery Routes
- Intranasal and oral routes, though less common in vaccine delivery, were as effective as intramuscular administration, offering alternative strategies for mass vaccination in outbreak scenarios.
Safety
- The vaccine showed no adverse effects across all tested macaques, underscoring its potential for human application.
- This research highlights the potential of mucosal and systemic vaccination strategies for Ebola virus, providing insights into immune mechanisms and paving the way for improved vaccine accessibility during outbreaks.
Cynomolgus 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
After 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.
Sources: Fuss et al. (2009), Reidhammer et al. (2016).
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.
Cynomolgus 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.
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?
We Ensure Quality and Quantity
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.
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
Razmara, A., et al. (2023). Single cell and genomic profiling of PBMC-expanded NK cells in first-in-dog clinical trial of allogeneic adoptive NK cell therapy for dogs with cancer. The Journal of Immunology, 210(1_Supplement): 224.05. DOI: 10.4049/jimmunol.210.Supp.224.05
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
