Kit for adipogenic differentiation

Mesenchymal stem cells (haHaMSCs) were obtained from adipose tissue of patients subjected to surgical operation. To obtain stromal cells, minced adipose tissue was digested with collagenase as described previously [17 (link)]. Immunophenotype and other characteristics of collected cells were described earlier [17 (link)]. HaMSCs (2278) were cultivated in a humidified atmosphere with 5% CO₂ in air at 37°C in AmnioMax C-100 Basal Medium (Gibco), containing AmnioMax Supplement C-100. Before treatments, cells were split no more than four times. Fluorescence-activated cell sorting analysis (FACS) has shown that the cultured HaMSCs did express MHC (major histocompatibility complex) molecules (HLA-ABC+) and adhesion molecules (CD44+, CD54 (low), CD90+, CD106+, CD29+, CD49b (low), and CD105); however, these cells were negative for hematopoietic markers (CD34-, CD45-, and HLA-DR-) and the marker CD117 [17 (link)]. In presence of an inducer (kit for adipogenic differentiation, “StemCell Technologies Inc.”), these cells underwent differentiation into adipocytes. HaMSCs were cultivated in the presence of DNA samples in a humidified atmosphere with 5% CO₂ in air at 37°C. Ethical approval for the use of haMSCs was obtained from the Regional Committees for Medical and Health Research Ethics (approval number 5).

MSCs were derived from adipose tissue of patients subjected to surgical operation. To obtain stromal cells, minced adipose tissue was digested with collagenase as described previously [23 (link)]. Tissue samples were mechanically disrupted in Dulbecco's Modified Eagle medium (DMEM) (Paneko, Moscow) containing 250 μg/ml gentamycin, 60 U/ml penicillin, and 60 U/ml streptomycin (Paneko). Cells were dissociated by incubation with 0.04% collagenase (Sigma) in DMEM with 10% fetal bovine serum (FBS) (PAA, Austria) at 37°C for 16 h. Cells were centrifuged at 200g for 10 min, transferred into slide flasks and cultivated at 37°C in AmnioMax Basal Medium with AmnioMax Supplement C100 (Gibco). Cultures were split no more than four times before experiments.
MSCs were characterized by standard markers using fluorescence-activated cell sorting (FACS): MHC molecules (HLA-ABC+) and adhesion molecules (CD44+, CD54 (low), CD90+, CD106+, CD29+, CD49b (low), and CD105); however, they were negative for hematopoietic markers (CD34−, CD45−, and HLA-DR−) and the marker CD117 (Dominici et al. 2006). Moreover, cells differentiated into adipocytes in the presence of inducers in a kit for adipogenic differentiation (STEMCELL Technologies). Ethical approval for the use of MSCs was obtained from the Regional Committees for Medical and Health Research Ethics (approval number 5).