Isolation, amplification and identification of mesenchymal stem cells de-rived from human adipose tissue

Currently, autologous and allogeneic adipose tissues represent a ubiqui-tous source of material for fat reconstructive therapies. However, these approaches are limited, and often accompanied by a 40-60% reduction in graft volume following transplantation, limited proliferative capacity of mature adipocytes for ex vivo expansion, and extensive adipocyte damage encountered when harvested with conventional liposuction techniques. Recently, cell-based approaches utilizing adipogenic progenitor cells for fat tissue engineering have been developed and were reported to promote both short-term in vivo adipogenesis and to repair defect sites. The aim of this study was to isolate stem cells from fat tissue than examine the growth of stem cells by invitro tests. For human adipose stem cell isolation (hASC), subcutaneous adipose tissue sites were obtained from female subjects undergoing elective procedures. Tissues were washed 3-4 times in phosphate buffered saline (PBS) and suspended in an equal volume of PBS supplemented with 1% FCS and 0.1% collagenase type I. The tissue was placed in an agitated water bath at 37 1C. The supernatant containing mature adipocytes, was aspirated. Portions of the SVF were suspended in DMEM medium. hASCs were selected based on their ability to adhere to tissue culture plastic and subsequently expanded to 75-90% confluence. Adipose stem cells were isolated and cultured on DMEM. To assess mesenchymal origin of stem cells we used flow-cytomery technique as well as differentiation to osteocyte and chondrocyte lines. The nature of the mesenchymal cells was confirmed by flow -cytometry tech-niques, based on the expression of CD90, CD105, CD166, and lack of expression of hematopoietic markers of CD34, CD31, and CD45. The successful differentiation of our stem cells to osteocyte, chondrocyte had been showed by specific Alizarin-Red and Toluidine-blue staining of cells. Although we have not the results of in vivo tests to support in vivo adipo-genesis either alone or in combination with natural or synthetic matrix, the results showed that stem cells isolation from adipose tissue was successful, and we provided an environment for differentiation of stem cells.