Towards standardised cryopreservation of equine mesenchymal stem/stromal cells: a multicentre comparative study

Mesenchymal stem/stromal cells (MSCs) are promising therapeutic tools to treat equine diseases, with particular interest for musculoskeletal injuries. Although MSCs have been extensively studied, cryopreservation protocols can vary substantially among laboratories, which may limit comparability and standardisation. The aim of this study was to evaluate the effect of different freezing media on equine MSCs and identify the cryopreservation conditions better suited to maintain high post-thaw viability and growth. The study was performed in parallel across three research centres to determine whether the protocols tested would yield comparable results under particular conditions of different labs. Three lines of equine bone marrow-derived MSCs from passages 3–5 were used in each lab, among those already available at their cell banks. Prior to the experiment, MSCs were expanded in standard culture conditions for one week. Cells were then frozen using nine freezing media, resulting from the combination of different proportions of foetal bovine serum (FBS, 90% 50%-10%), and dimethyl sulfoxide (DMSO, 10%-7.5%-5%), using Dulbecco’s Modified Eagle Medium (DMEM) as basal medium. One month after freezing, all samples were thawed and cell viability was assessed. Cells were subsequently reseeded, cultured for an additional week, and counted to evaluate post-thaw growth and expansion capacity. By comparing a range of freezing conditions under harmonized experimental procedures, this multicentre study generates information on the influence of serum and DMSO concentrations on equine MSC performance. The findings support the optimization of cell banking procedures and may contribute to the development of standardised cryopreservation protocols for research, translational studies, and clinical applications.