Cytotherapy, 2010; 12: 96–106 Correspondence: Patrick C. Baer, Goethe University, Department of Internal Medicine III, Division of Nephrology,Theodor-Stern-Kai 7, 60590 Frankfurt/M, Germany. E-mail: P.Baer@em.uni-frankfurt.de (Received 7 August 2009; accepted 28 September 2009) ISSN 1465–3249 print/ISSN 1477–2566 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS) DOI: 10.3109/14653240903377045 Human adipose-derived mesenchymal stem cells in vitro: evaluation of an optimal expansion medium preserving stemness PATRICK C. BAER 1 , NADINE GRIESCHE 1 , WERNER LUTTMANN 2 , RALF SCHUBERT 3 , ARLETTE LUTTMANN 2 AND HELMUT GEIGER 1 1 Division of Nephrology, Department of Internal Medicine III, Goethe University, Frankfurt, Germany, 2 EuroBioSciences GmbH, Friesoythe, Germany, and 3 Department of Pediatrics, Goethe University, Frankfurt, Germany Background aims. The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell thera- peutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintain- ing the undifferentiated state has not been described. Methods. We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. Results. The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. Conclusions. Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential. Key Words: adipose tissue, culture, differentiation, expansion, medium, mesenchymal stromal cells Introduction Adipose-derived mesenchymal stromal cells (ASC) are multipotent adult mesenchymal stromal cells (MSC) that have characteristics very similar to bone marrow (BM)-derived mesenchymal stromal cells (BMSC) (1–5). Compared with BMSC, ASC are easier to isolate because cell aspiration does not require painful procedures or cause donor site injury, and adipose tissue contains large numbers of stem cells compared with BM. At present, the cells are defined by plastic adhesion and their immunophe- notypic profile, cell morphology and multilineage differentiation potential (3,6,7). However, cultured ASC (as MSC in general) seem to be a heteroge- neous cell population (8) and no marker specifically and unequivocally identifies undifferentiated ASC. Several studies have shown that ASC possess the potential to differentiate into multiple cell types of mesodermal, endodermal and epidermal origin, i.e. chondrocytes, osteoblasts, fibroblasts, adipocytes, smooth muscle cells, neurons, cardiomyocytes, hepa- tocytes and endothelial cells (2,3,7). We have shown previously that ASC undergo differentiation towards the epithelial lineage when treated with all-trans retinoic acid (ATRA) (9) or conditioned medium from primary renal tubular epithelial cells (10). This versatile differentiation potential places ASC at the forefront of stem cell-based therapies and transplan- tation, and may therefore become an emerging and novel organ regeneration tool for the treatment of damaged tissues (11). Nevertheless, the use of cul- tured ASC in stem cell-based therapies is impeded by limitations of proliferation/population doubling and loss of stem cell properties during ex vivo expansion, probably because of the culture medium or supple- ments used. Although non-expanded mesenchymal stem cells have also been used in cell therapy (12), the prerequisite to obtain large cell numbers for treatments is to expand ASC in vitro by maintain- ing their stem cell nature. Expansion protocols in