Horses in Biomedical Research
Horses have historically played pivotal roles in medical breakthroughs. For example, these animals have been instrumental in the development of antitoxins for diseases like diphtheria. The use of horse serum in particular is recognized for providing a way to create life-saving treatments and vaccines. Due to their large size, horses enable the collection of significant amounts of blood and serum for the production of biologics and therapeutics. One of the known examples of these is equine chorionic gonadotropin (eCG), which is derived from pregnant mare serum and widely used to enhance fertility in livestock.
Advantages of Horse PBMCs
Horse peripheral blood mononuclear cells (PBMCs) offer several advantages in biomedical research, particularly in the study of specific diseases and formulation of therapeutic interventions.
Non-Invasive Collection
PBMCs from horses can be collected in a relatively non-invasive method. Due to their large size, horses can be easily sourced for blood through the jugular venipuncture. This is minimally stressful compared to other methods and also allows for repeated sampling.
Rich Source of Stem Cells
Equine PBMCs are an excellent source of multipotent mesenchymal stromal cells (MSCs). MSCs have displayed great therapeutic potential in regenerative medicine due to their potential to differentiate into different cell types such as osteocytes, adipocytes, and chondrocytes – a trait highly useful for treating musculoskeletal injuries and other disorders.
Immunomodulatory Properties
PBMCs demonstrate immunomodulatory activities that can help manage inflammatory conditions. PBMCs from horse have been shown to inhibit proliferation of lymphocytes and promote the production of IL-10 and other anti-inflammatory cytokines that aid in inflammation reduction and healing.
Low Immunogenicity
Equine PBMCs have been found to produce low levels of Major Histocompatibility Complex (MHC), a trait that is advantageous for cell-based therapy development.
Clinical Potential
everal studies indicate that horse PBMCs are showing promise in the treatment of osteoarthritis and tendon injuries. PBMC-derived autologous MSC therapy, for instance, have been associated with alleviation of musculoskeletal disorders.
Sources: Koerner et al. (2011), McCarthy et al. (2019), Smith et al. (2023), Ahern et al. (2024)
Horse 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.
Horse 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)
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.
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 Study that Used Horse PBMCs
The study by McCarthy et al. (2019) titled “Peripheral blood-derived mesenchymal stem cells for autologous cell therapy in horses: A study on efficacy and safety” investigates the use of equine peripheral blood mononuclear cells (PBMCs) as material for deriving mesenchymal stem cells (MSCs) in order to develop autologous cell therapy in horses.
Key Highlights of Horse PBMCs
Objective
- The aim of the study was to determine whether MSCs derived from PBMCs could be harvested, expanded in vitro, and safely administered to young and aged lame horses in order to assess the effectiveness of these cells across the subjects.
Methodology
- PBMCs were isolated from the blood of 29 young and aged horses. The cells were then expanded in vitro to generate MSCs. To confirm their identity, MSCs were characterized phenotypically using flow cytometry. The expanded MSCs were administered intravenously at a dose of 50 million cells to 24 horses. The efficacy of the MSC treatment was assessed using the American Association of Equine Practitioners (AAEP) lameness scale – a standardized method for evaluating lameness improvement in horses. Safety was assessed through intradermal immune reaction testing for immune reactions and post-treatment monitoring for adverse effects.
Results
- The treatment showed significant improvements in lameness scores among the treated horses, indicating that PBMC-derived MSCs effectively alleviated lameness associated with orthopedic conditions. The MSCs demonstrated immunomodulatory activities through the inhibition of lymphocyte proliferation and induction of interleukin-10 (IL-10) production. This result indicates that PBMC-derived MSCs can potentially influence immune response modulation, which can be beneficial in managing inflammatory conditions.
- PBMC-derived MSCs are deemed safe for use in equine patients due to the absence of immediate and delayed immune reactions as well as the low expression of Major Histocompatibility Complex (MHC) class I and II markers, indicating low immunogenicity and suitability of MSCs for autologous therapies.
Conclusion
- The study concluded that equine PBMC-derived MSCs can be effectively isolated, expanded, and administered as a safe and efficacious treatment for lameness in horses. This research highlights the potential of using PBMCs as a viable source of stem cells for regenerative therapies in veterinary medicine. PBMC-based MSCs thus offer a less invasive alternative compared to traditional treatment options.
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
