The Effect of a 7 Year-Long Cryopreservation on Stemness Features of Canine Adipose-Derived Mesenchymal Stem Cells (cAD-MSC)

SIMPLE SUMMARY: The use of canine adipose tissue-derived mesenchymal stem cells represents a promising tool in the emerging field of autologous cell therapy in veterinary medicine. Cells need to be isolated and expanded in vitro in order to obtain the sufficient amount for clinical application, but long-term cultivation before therapeutic use is not recommended, since the cells may lose their stemness features. Alternatively, the cells can be cryopreserved and used as needed and in a short time after thawing. This study evaluated the effect of a 7 year-long cryopreservation using 10% dimethyl sulfoxide with different fetal bovine serum concentrations (from 10 to 90%) on different cell passages. The aim was to establish the most appropriate cell passage and serum percentage for the long-term cryopreservation of cells ensuring the maintenance of the stemness features. Cells were expanded in vitro from P0 to P1–P2 passages and subsequently frozen. This study demonstrated that a high percentage of serum (80%) is necessary to obtain optimal cryopreservation with 10% DMSO. Cells thawed at passages from P2 to P4, even after seven years, could be considered in the studies on therapeutic application and in the in vitro study, because they maintain stem potential after cryopreservation. ABSTRACT: Mesenchymal stem cells (MSCs) are used in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. Their clinical application requires a ready off-the-shelf amount of viable therapeutics doses. For this purpose, it is useful to cryopreserve MSCs to gain a ready and controlled source of abundant autologous stem cells. We evaluated the effect of 7 years cryopreservation using 10% dimethyl sulfoxide (DMSO) with different fetal bovine serum (FBS) concentrations (from 10 to 90%) on different passages of MSCs isolated from canine adipose tissue (cAD-MSCs). The study aimed to evaluate the most adequate cell passage and FBS percentage for the long-term cryopreservation of cells by maintaining the stemness features. Phenotype morphology, cell viability, osteogenic and adipogenic differentiation potentials, proliferative potential and expression of pluripotency markers were analyzed in thawed cells and compared with fresh ones. We demonstrated that cells cryopreserved with at least 80% FBS maintain unaltered the stemness characteristics of the freshly isolated cells. In particular, cells of P0–P1 passages have to be expanded in vitro and subsequently cryopreserved and cells of P2–P4 passages should be considered in the studies on therapeutic application and in vitro study of cAD-MSCs.