Advantages of Beagle PBMCs
In the 1950s and 1960s, extensive use of beagles in toxicology studies, particularly in assessing the effects of pharmaceuticals and environmental exposures, started. Since then, the breed became widely recognized as a standard test subject for new drugs due to its genetic and physiological characteristics that allowed data to translate effectively to human outcomes.
Beagle PBMCs are instrumental in clinical research because they provide a consistent and ethical source of immune cells that closely resemble human immune function which is helpful when studying immune response, developing therapeutic measures and evaluating drug safety. These are the advantages of using beagle PBMCs.
Genetic Similarity to Humans
Their genetic and physiological similarities with humans make beagle PBMCs ideal for investigating human-like immune behavior especially in preclinical studies.
Consistent Immune Profile with Standardized Protocols
The immune profile of beagles have been well characterized so results are predictable and reproducible. Experimental protocols have also been standardized making results more reliable.
Reliable Cryopreservation and Viability
Beagle PBMCs respond to cryopreservation well so cells can be stored for future studies or experiment replication. Post-thawing viability of beagle PBMCs is also high, increasing the reliability of in vitro assays.
Comparable to Non-Human Primate (NHP) Models
PBMCs from beagles have been used as a complementary model when non-human primates cannot be used due to ethical or financial restrictions.
Autoimmune and Inflammatory Disorders
Beagles are known to be susceptible to specific autoimmune diseases so their PBMCs offer a good opportunity to study the mechanisms behind these conditions.
Ethical and Consistent Supply
Beagles are widely used in research, thus beagle PBMCs offer an ethical alternative to intrusive whole-animal experiments. Since beagles are easy to collect samples from due to their calm temperament, a steady supply of PBMCs for long-term studies is an advantage.
Beagle 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.
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.
Beagle 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)
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?
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?
Published Research Using Beagle PBMCs
The study by Razmara et al. (2023) investigates the use of peripheral blood mononuclear cells (PBMCs) from beagle donors in a clinical trial for allogeneic adoptive natural killer (NK) cell therapy targeting dogs with naturally occurring melanoma.
Key Highlights of Beagle PBMCs
Cell Expansion and Function
- PBMCs were used as a source for NK cell expansion without prior CD5 depletion, yielding robust NK cells with enhanced activation and cytotoxicity-related gene expression (e.g., GZMB, NCR3). This approach showed improved NK cell functionality compared to traditional methods.
Clinical Outcomes
- The treatment was safe, with no significant adverse effects observed. A notable case reported a dog surviving 445 days post-treatment, indicating potential efficacy of the therapy.
Genomic Insights
- Single-cell sequencing revealed sustained NK cell activity and uncovered previously unrecognized similarities between NK and CD8 T cell gene expression. This suggests potential new biomarkers and mechanisms underlying immune responses in canine models.
Implications for Immunotherapy:
- The study highlights PBMC-expanded NK cells as a promising avenue for cancer immunotherapy in veterinary and potentially translational contexts. It also underscores the value of genomic profiling in optimizing cell-based therapies.
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
