In vitro isolation and characterization of Mesenchymal Stem Cells (MSCs) from lipoaspirate: a Preclinical Protocol

Mesenchymal Stem cells (MSCs) could provide alternative therapeutic solutions for a number of bone and cartilage tissue diseases, playing an important role in regenerative medicine. Many sources of stem cells are available and classical research has focused on MSCs isolated from bone marrow. However, procurement of such cells is a painful procedure for patients and yields low numbers of harvested cells. On the other side, adipose stem cells (ASCs) are easily obtainable in large quantities trough a minimally invasive procedure, Liposuction, with a little patient discomfort and, for this reason, the use of lipoaspirate may provide an alternative source of stem cells. Adipose-derived stem cells (ADSCs) have multiple properties, including self-renewal and pluripotent ability to differentiate into several mesodermal lineages. In previous studies, ADSCs showed chondrogenic and osteogenic differentiation potential, becoming of increased interest in orthopaedic surgery. An essential function of ADSCs is the production and secretion of growth factors and angiogenic factors that might influence surrounding cells, especially if cultured under in vitro hypoxic conditions. Finally, ADSCs produce several immunomodulatory cytokines, potentially useful in the treatment of several degenerative diseases. The aim of this study was to present a new “in vitro” ADSCs isolation and characterization protocol: after obtaining fat tissue from donors lipoaspirate, we compared the actual gold standard isolation method, enzymatic digestion, with a new isolation method, mechanical isolation, to verify if the extracted ADSCs hold and preserve the characteristics of adult stem cells. Methods: Lipoaspirate from forty human donors were obtained by minimally invasive mini-liposuction procedures: by definition, lipoaspirate contains a relatively high content of ASCs (1-5% of isolated nucleated cells). A minimum starting volume of 30 mL of lipoaspirate was obtained in all procedures for a sufficient yield of mesenchymal stem cells (MSCs). Lipoaspirate was stored at 2-8º C for a maximum of 24 hours before use. Tissue from each donor was split in two samples and each treated by two different in vitro procedures with the goal to isolate ADSCs: enzymatic digestion and experimental mechanical isolation procedure. Tissue from both methods were first diluted with an equal volume of phosphate-buffered saline (PBS), centrifuged at 500xg at 22° for 5 minutes, digested adding 1 ml of collagenase digestion solution for every 1 ml volume of tissue, incubated at 37ºC in a pre-warmed orbital shaker for 30 minutes, centrifuged at 22°C for 5 minutes at 500xg and finally suspended for expansion in alpha-MEM medium adding 10% of bovine fetal serum. Subsequently, MSCs were characterized following the guidelines of the Society for Cellular Therapy (SCT). Results: Characterization of the processed tissue was performed after digestion and “in vitro” culture expansion of plastic-adherent cells. Our results showed that cells from both study groups were adherent to the plastic having fibroblast morphology. The cells were immune-characterized by Flow cytometry. Results showed a phenotipic mutation during the process of cellular expansion, from the isolation of the stromal vascular fraction to the selection of a purely population with mesenchymal stem cells properties in the subsequent passages. After the isolation of MSCs, the obtained ADSC showed a subpopulation (immunophenotype differentiation) expressing specific markers such us CD 34, a marker associated with the state of vascular and endhotelial progenitor, and CD 146, a pericytic markers. Finally, the evaluation of the cells differentiation potential showed that ADSCs, under controlled condition, were able to differentiate into adipogenic, chondrogenic and osteogenic cells. The results were similar for both differential methods. Conclusion: Our experience suggests that ADSCs enzymatically and mechanically isolated from Lipoaspirate respond to the three criteria of the Society for Cellular Therapy (SCT) and, for this reason, can be considered as MSCs. New methods to treat fat tissue and obtain ADSCs, as the mechanical method described in this article, can be safely developed to isolate in an easier way these promising cells. Therefore, improved characterization protocols are still necessary in order to make a final population of ADSCs as an homogeneous cellular product ready for clinical application in the field of osteogenic and chondrogenic tissue engineering. Finally, more studies are necessary to understand the immunomodulatory and anti-inflammatory properties of ADSCs before clinical applications to treat different orthopaedic pathologies.