**6. Isolation of MSCs from adipose tissue**

The isolation of rat mature adipocytes and adipose tissue progenitor cells was described in literature for the first time by Rodbell [41]. The protocol was based on the fragmentation of adipose tissue into small portions, followed by enzymatic digestion with collagenase type I at 37°C and the subsequent centrifugation to separate the different cell fractions. The obtained supernatant was composed of mature adipocytes and the pellet fraction consisted of the stromal vascular fraction (SVF) components, which comprise a heterogeneous cell population, including circulating blood cells, fibroblasts, pericytes, and endothelial cells, as well as "pre‐ adipocytes" or adipocyte progenitors. Stem cells and progenitor cells represent about the 3% of all cell populations [42]. Stem cells derived from the adipose tissue (ASCs) represent a purified population of the adherent stem cells present in the adipose tissue, since all other cell types are removed or die with time. Currently, ASC recovery is quick and easy to perform from the subcutaneous adipose tissue, as it could be successfully collected via lipectomy or from the tail base in horses and from the inguinal region [43] or during ovariohisterectomies in dogs and cats. Stem cells derived from the adipose tissue have been increasingly used for cell therapy both in humans and animals [44], either as freshly isolated, SVF cells, or as cultivated ASCs [43]. ASCs proliferate rapidly with a high cellular activity, making them an ideal source to obtain MSCs [45]. The most important advantage of adipose‐derived stem cells is their abundance: from 1 g of adipose tissue an average of 0.5–2.0 × 106 SVF cells can be isolated, which gives 1–10% of stem cell yield [46]; in comparison, MSCs constitute only 0.001–0.01% of BM [15]. When autologous ASCs are used, the adipose tissue is collected 2 or 3 weeks before the treatment and the animal receives the cultivated cells, but long‐term cultivation of ASCs before therapeutic use is not recommended, since the cells may lose their progenitor charac‐ teristics [47]. The use of allogeneic ASCs has been also performed; since these cells have immunoregulatory properties [48], this approach would allow the use of species‐specific allogeneic cryopreserved cells, avoiding the need for collection of tissue from the patient [49].
