The Stromal Vascular Fraction from Canine Adipose Tissue Contains Mesenchymal Stromal Cell Subpopulations That Show Time-Dependent Adhesion to Cell Culture Plastic Vessels

SIMPLE SUMMARY: Adipose tissue-derived mesenchymal stromal cells (MSCs) are stem-like cells extensively studied for regenerative applications in both human and veterinary medicine. Their isolation is usually performed by enzymatic digestion of adipose tissue followed by cell seeding. Tissue remnants that are still floating 48 h after seeding are discarded. We observed that waste tissue fragments still contain cells that adhere belatedly to the dish. In this study, we aimed to investigate their basic properties to speculate on the possible existence of MSC subpopulations. Samples of subcutaneous adipose tissue from three dogs were enzymatically digested. We obtained three cell populations that adhered to the culture dish 48, 96, and 144 h after seeding. Cells were analyzed through different methods to assess possible differences in phenotype, viability, proliferation, and differentiation ability. No significant differences were found between the three subpopulations. However, this procedure has proven to be a valuable method for dramatically improving MSC yield. As a consequence of cell recovery optimization, the amount of tissue harvested could be reduced, and the time required to obtain sufficient cells for clinical applications could be shortened. Nevertheless, functional differences cannot be excluded, and other assays are needed to investigate possible different biological properties. ABSTRACT: Adipose-derived mesenchymal stromal cells (MSCs) are extensively studied in both human and veterinary medicine. Their isolation is usually performed by collagenase digestion followed by filtration and removal of nonadherent tissue remnants 48 h after seeding. We observed that waste tissue fragments contain cells that adhere belatedly to the plastic. We aimed to investigate their basic properties to speculate on the possible existence of MSC subpopulations. Adipose tissue from three dogs was enzymatically digested. Three cell populations that adhered to the culture plastic 48, 96, and 144 h after seeding were obtained. After expansion, they were analyzed by flow cytometry for MSC-positive (CD90, CD44, and CD29) and -negative (CD14, MHCII, and CD45) markers as well as for endothelial, pericyte, and smooth muscle cell markers (CD31, CD146, and alpha-SMA). Furthermore, cells were assessed for viability, doubling time, and trilineage differentiation ability. No significant differences were found between the three subpopulations. As a result, this procedure has proven to be a valuable method for dramatically improving MSCs yield. As a consequence of cell recovery optimization, the amount of tissue harvested could be reduced, and the time required to obtain sufficient cells for clinical applications could be shortened. Further studies are needed to uncover possible different functional properties.