Preparation of adipose tissue stromal vascular fraction by mechanical digestion for point of care application in veterinary medicine.

Preparation of adipose tissue stromal vascular fraction by mechanical digestion for point of care application in veterinary medicine. OBJECTIVES: Mesenchymal Stem Cells (MSCs) derived from adipose tissue have a growing role in the therapy of a variety of animals diseases, including wound healing, tendonitis, bone defects, osteoarticular pathologies and neurodegenerative diseases. As a matter of fact, fat tissue represents a complex source of cells as well as bioactive factors able to contribute to the healing of injured tissues. The term Stromal Vascular Fraction (SVF) has been introduced in recent years to describe the mixed cell population (endothelial cells, pericytes, smooth muscle cells, stromal progenitor cells) isolated from fat tissue. We evaluated the feasibility of a simple procedure for the preparation of a SVF derived from fat in the dog, to be used “point of care” ”, i.e. shortly after the collection of the biological sample and after a minimal manipulation. A key concept of the approach we propose, is that fat tissue is a source of MSCs but also of other cell types able, for example, to promote the formation of blood vessels and to modulate the different phases of tissue healing. MATERIALS AND METHODS: SVF was prepared by mechanical treatment of 2 grams of subcutaneous and visceral fat. A mechanical digestion was performed inside a 50 ml test tube using a set of 6 mm diameter sterile steel balls obtaining tissue fragments ranging from 1 mm3 to 4 mm3. The tissue fragments were cultured either on plastic surface or inside a three-dimensional scaffold prepared with Platelet Rich Plasma, thrombin and culture medium. Cell cultures were maintained until passage four for cells grown on plastic surface, or for 10-12 days for cells grown inside the three-dimensional matrix. RNA was collected to analyze the expression of markers typical for MSCs as well as endothelial cells and pericytes. RESULTS: The fragments released in the surrounding environment a high number of actively proliferating cells that populated the plastic surface or the three-dimensional gel in different culture conditions. Noteworthy, complex multicellular structures connecting different tissue fragments were often observed, thus suggesting the ability of the cells to respond to chemotactic signaling. The histological examination of the cultured fragments demonstrated that the tissue maintained its structural organization for at least 10-12 days. In addition, the cells released by tissue fragments adhered to the culture plate and were able to differentiate into adipocytes and osteoblasts. RT- PCR analysis demonstrated that cells expressing phenotypes of MSCs and endothelial cells are present in cell cultures. CONCLUSIONS: Although incomplete, the preliminary characterization of the cells obtained by the in-vitro culture of SVF, let us hypothesize that the cell population include MSCs maintaining their typical features. The safeguard of the tissue architecture and the long-lasting maintenance of its vitality guarantee a positive evaluation of the efficacy and efficiency of the proposed mechanical treatment.