INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 6, ISSUE 05, MAY 2017 ISSN 2277-8616 16 IJSTR©2017 www.ijstr.org Characterization Of Bovine Adipose-Derived Stem Cells Daniel Cebo ABSTRACT: Bovine adipose-derived stem cells were obtained from the subcutaneous abdominal adipose tissue. The cells were cultured by the modified tissue-explants method developed in our laboratory, and then analyzed using optical microscopy and flow cytometry. These cells were able to replicate in our cell culture conditions. cell Flow cytometry showed that bovine adipose-derived stem cells expressed mesenchymal stem cell markers (CD73 and CD90). Meanwhile, haematopoietic markers (CD45 and CD34) are absent from bovine adipose-derived stem cells. We also induced their adipogenic differentiation in vitro. KEYWORDS: adipocytes, adult stem cells, mesenchymal stem cells, regenerative medicine ———————————————————— 1. INTRODUCTION Adipose tissue is an abundant source of mesenchymal stem cells, which have shown promise in the field of regenerative medicine [1]. There is a growing scientific interest in the plasticity and therapeutic potential of adipose-derived stem cells (ASCs). ASCs are multipotent, differentiating along the adipocyte, chondrocyte, myocyte, neuronal, and osteoblast lineages [2]. ASCs have potential applications for the repair and regeneration of acute and chronically damaged tissues [3]. While embryonic stem cells (ESCs) exhibit unlimited differentiation potential, the application of ESCs in cell-based therapies is limited by ethical and legal issues. Mesenchymal stem cells (MSCs) especially cells derived from adipose tissue also show great differentiation potential. ASCs have been used in studies of osteoarthritis, diabetes mellitus, heart disease, and soft tissue regeneration and reconstruction after mastectomy and facial repair [4]. Many kidney diseases are associated with inflammation and altered immune response. ASCs are known for their anti-inflammatory properties and immune modulation. Demonstration that the phenotype and immunosuppressive ability of ASCs are not affected by human kidney disease could have clinical significance [5, 6]. In order to be able to obtain ASCs cell preparations suitable for basic investigations as well as for development of future therapeutic protocols, it is important that the critical isolation steps are properly carried out. Here, we describe the isolation of ASCs using a tissue explants-based procedure from the bovine subcutaneous fat tissue that is also adaptable to several animal species. The aim of this study was to isolate, cultivate and identify adipose-derived stem cells. 2. MATERIALS AND METHODS 2.1. Materials Dexamethasone, 3-isobutyl-1-methylxanthine, insulin, Dulbecco’s modified Eagle’s medium (DMEM/F12), DMEM/Ham’s nutrient mixture F12, Hanks’ balanced salt solution (HBSS), and 100× antibiotic-antimycotic solution were all purchased from Sigma-Aldrich (St. Louis, MO). Foetal bovine serum was purchased from Atlanta Biological (Lawrenceville, GA). Trypsin-EDTA was purchased from Mediatech Inc. (Herndon, VA). Cell markers: fluorescein isothiocyanate (FITC), allophycocyanin (APC) or phycoerythrin (PE) are purchased from BD Biosciences, USA. 2.2. Animals Adipose tissue was collected from beef cattle slaughtered at a local abattoir. Subcutaneous adipose tissue was transported to the laboratory in cold HBSS supplemented with 10% antibiotic-antimycotic solution. Tissues were collected post-mortem from animals slaughtered for food and not for research; therefore, Institutional Animal Care and Use Committee approval was not required 2.3. Culture and expansion Tissue was extensively washed with phosphate buffered saline (PBS) to remove contaminating debris. After removing excess water, the samples were minced into very small pieces (less than 5 mm) with scissors and placed in tissue culture flasks under sterile conditions. The spacing between adjacent tissues was around 8-10 mm. Then the flask was tipped up on its side, with the cap loosened by 1/4 turn to allow CO 2 atmosphere exchange, and the tissue was let to “dry” at the bottom of the flask for 1-2 hours. The flask was gently laid back down, allowing media to again surround and cover the tissue. After the explants adhered to the bottom, the flask was gently laid back down, allowing growth medium containing D-MEM (without FCS) to surround and cover the tissue. The flasks were maintained in a humidified atmosphere of 5% CO 2 at 37 °C and the medium was changed every day. On the day 7, medium was changed to D-MEM with 20% FCS. Medium was changed every second day. On the day 14, medium was changed to D-MEM with 10% FCS. When the cells grew to 80% confluence they were passaged using standard trypsinization techniques cells were counted using the Trypan Blue exclusion test (haemocytometer). 1.0 x 10 6 __________________________  Daniel Cebo  Institute of Veterinary Physiology, Free University of Berlin, Oertzenweg 19b, 14163 Berlin, Germany  dcebo78@gmail.com