Validation of equine cell lines as a cellular replacement (3Rs) to model equine primary cells for in-vitro studies


Norman Hayward Fund



Research Period


Area of study



Principal investigator: Dr. Maria R. Lopez-Alvarez


Equine Immunology is a fast growing and complex field with significant ongoing research. Increased understanding of how the immune system fights infectious disease will help to develop new treatments modulating the immune response and enhancing the protection of horses against infectious challenge. Alternative approaches allowing the reduction and replacement of animals are essential in research according to the 3Rs principles.
Finding and validating alternative systems to in-vivo studies or samples obtained from animals should be considered to perform high quality research. The results obtained from the validation of existing equine cell lines as substitutes for primary cells will significantly broaden their use as an in-vitro alternative to study equine immunity and contribute to the welfare of animals. Ultimately, the wider use of these cell lines will help to develop new therapies and/or preventive strategies to fight equine infectious diseases, reducing the use of primary cells from animals in research.

Project summary:

Two cell lines, e-CAS (equine macrophages) and eqT8888 (equine lymphocytes) were assessed to establish their suitability as replacement for equine primary cells in in vitro studies about the immune response.
During this project, aliquots of e-CAS cells stored at the Animal Health Trust were found to contain mouse macrophages instead of equine cells. Nevertheless and as cell lines from different species have been previously considered as replacement of primary cells, the ability of e-CAS to phagocyte and to migrate to stimulants, as well as their antimicrobial activity through the production of nitric oxide, were assessed and compared to those of equine primary phagocytes. Results indicated a level of translatability in terms of phagocytosis and release of nitric oxide. However, there were differences in migratory profiles between the two cell types, with equine primary cells showing a significantly more pronounced migration towards host-restricted pathogen (Streptococcus equi) supernatants when compared to general stimulants.
On the other hand, eqT8888 cells were confirmed as equine cells but their characterisation using cellular and molecular approaches was not conclusive and, therefore, they could not be considered as a suitable replacement for equine primary cells.
In conclusion, our data show that e-CAS could be used as a replacement for equine primary neutrophils to preliminary assess phagocytosis and nitric oxide release in vitro when the effect of general stimulants is evaluated. This will contribute to the reduction of samples taken from animals, in line with the 3Rs principles. However, as the innate immune system seems to be more specific than previously thought, particularly when considering host-restricted pathogens, results obtained from experiments using e-CAS should be finally confirmed in equine cells.