Isolation and characterization of canine umbilical cord mesenchymal/stromal stem cells

Mesenchymal stem cells (MSCs) have therapeutic potential due to their immunomodulatory and anti-inflammatory properties. In veterinary medicine, adipose tissue is the most common source of MSCs to treat canine disease, but the collection process is invasive, and the cells are influenced by the age and health conditions of the donor. These problems enhance interest in seeking alternative MSC sources, such as perinatal tissues. In this study, we developed and validated an optimized protocol for isolating canine umbilical cord MSCs for application in veterinary therapies. Umbilical cords obtained from cesarean sections were processed using three different protocols, involving combinations of mechanical and enzymatic tissue dissociation. The cells were cultured and evaluated for membrane receptors by flow cytometry to identify MSCs and assessed for their differentiation capacity. The number of cells obtained did not differ significantly between the combined protocol with trypsin and collagenase (TRIP + COL) and the collagenase protocol (COL). In in vitro culture, the combined TRIP + COL and COL yielded 12 to 14 times more cells, respectively, in the first passage than the explant (EXP) group, within fewer days of culture. Additionally, the cells obtained from these protocols showed a greater capacity for expansion over passages, and cells from both protocols showed fibroblast-like morphology and proliferation capacity up to the sixth passage. The cells obtained from these protocols were characterized by phenotype: CD45^-, CD34^-, CD14^-, HLA-DR^-, CD29⁺, CD44⁺, and CD90⁺, consistent with MSC identity. However, CD90 expression in the cells decreased significantly at sixth passage. Regarding differentiation, cells obtained from the COL protocol showed a capacity for commitment to the chondrogenic and osteogenic lineages. In conclusion, the COL and TRIP + COL protocols were more effective than the EXP protocol in terms of both the number and quality of isolated cells. However, due to its less-aggressive enzymatic nature, we considered the COL protocol to be the best method to obtain canine MSCs.