Immunophenotypic analysis of human adipose-derived stem cells through multi-color flow cytometry

Nowadays, it is established in the scientific literature that adipose tissue is highly bioactive, and it is at the center of regenerative medicine and tissue engineering. Adipose-derived stem cells (ASCs) are regarded as one of the most promising sources of adult or somatic stem cells, identified thus far for cell therapy and regenerative medicine across a wide range of diseases (1-3). ASCs are included in and isolated from the adipose tissue’s stromal vascular fraction (SVF), which consists of a heterogeneous mesenchymal cell array, including not only ASCs but also preadipocytes, fibroblastic cells, endothelial cells, pericytes, vascular smooth muscle cells, hematopoietic cells (4,5), macrophages, and lymphocytes (6). It is also largely recognized by the researchers the necessity to establish a common isolation method and a standard characterization of human stromal and stem cells from SVF and ASCs to facilitate the comparison of the study outcomes and so the clinical research advancement within the field of adipose science. In this study, we proposed an innovative and non-enzymatic protocol to collect clinically useful ASCs, within freshly isolated SVF from adipose tissue, by centrifugation of the infranatant portion of lipoaspirate in order to determine the characteristic cytofluorimetric pattern prior to in vitro culture. Thirty-two patients affected by dry age-related macular degeneration with a mean age of 69.4 years, after having read and signed the informed consent, were recruited taking into account inclusion and exclusion criteria according to the study protocol of the Ethical Committee. With the patients under local anesthesia, adipose tissue was suctioned and the SVF was isolated according to the Lawrence and Coleman technique (7-10). Characterization of the cell composition of SVF has been accomplished through multi-color flow cytometry (CytoFLEX Flow Cytometer, Beckman Coulter, Brea, CA, USA) that allows the identification of the surface marker expression of the cells in vitro. The panel of cell surface antigens was chosen in agreement with the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy recommendations (3). The immunophenotypic analysis was performed to confirm the mesenchymal nature of isolated cells. The following fluorochrome-labeled monoclonal antibodies were used: CD31-PE, CD34-PC, and CD45-APC. The markers were used in combination with ViaKrome, which determines cell viability, excluding debris and dead cells induced by the isolation protocol. Cells were incubated with specific mAbs for 15 min. The software CytEpert Version 2.2.0.97, CytoFLEX (Beckman Coulter) was used to create dot plots and to calculate the cell composition percentages according to the profile of the surface marker expression. The SVF yielded a mean of 73.32%±10.89% cell viability evaluated with CALCEINA-FITC, i.e., cell-permeant dye. ASCs were positive for PC7-labeled mAb anti-CD34 and negative for both PE-labeled mAb anti-CD31 and APC-labeled mAb anti-CD45. The frequency of ASCs, estimated according to the panel of cell surface markers used, was 51.06%±5.26% versus the unstained ASCs subpopulation which was 0.74%±0.84% (P<0.0001). The ASCs events/µL were 1,602.13±731.87/µL (Appendix 1). The findings suggested that ASCs found in freshly isolated adipose SVF obtained by centrifugation of lipoaspirate can be immunophenotypically identified with a basic panel of cell surface markers. Therefore, we used a combination of positive and negative markers in the same multiparameter flow cytometric analysis to phenotype the cells and one viability dye to exclude apoptotic cells and other debris, indicating the viability of ≥70% for ASCs, according to IFATS recommendations. To date, it is challenging to define a common isolation method and characterization profile of ASC found in adipose tissue (11,12). To the best of our knowledge, we proposed a basic cytofluorimetric pattern of in vivo ASC population found in freshly isolated SVF obtained by centrifugation of the infranatant portion of lipoaspirate from adipose tissue without purifying them by adherent culture isolation and expansion in vitro. This paper has limitations and is only an indication for future studies. The flow cytometry pattern of SVF includes several clusters of differentiation (CD) that need to be analyzed. Indeed, CD34 alone is not an indication of SVF stem cell positivity and CD90, CD73, CD105, and CD275 should be associated. Furthermore, to be sure of having identified the SVF cells, these should be able to differentiate in an osteogenic, chondrogenic, and adipogenic sense.