HUMAN MESENCHYMAL STEM CELLS AS BASIC TOOLS FOR TISSUE ENGINEERING: ISOLATION AND CULTURE

摘要

Stem cells are immature cells capable of autoreplication, which are able to generate various mature cell types and have a remarkable viability and proliferative capacity. Mesenchymal stem cells (MSC), under proper conditions, can give rise to osteoblasts, adipocytes, chondrocytes, myocytes and even neurons. Several procedures applied in tissue engineering imply harvesting autologous MSC, expanding them in culture without loss of stemness, inducing differentiation, seeding them on suitable scaffolds in accordance with the targeted tissue type and implanting the construct into the patient’s body. During this study, bone marrow has been extracted from 9 patients by aspiration from the upper posterior iliac crest and used in order to isolate the MSC by employing three different methods: (1) the Ficoll-Paque technique for the isolation of mononucleated cells followed by the separation of MSC by adherence to plastic, (2) Ficoll-Paque followed by the immuno-magnetic separation of MSC using a CliniMACS system and (3) a negative selection procedure of MSC using the RosetteSep technique followed by adherence to plastic. We have prepared and optimized our media, assuring control and reproducibility of the results. Finally, an immunophenotypic characterization of the cells by flowcytometry has been performed. On the 3rd day after seeding the isolated mononucleated fraction in the optimally prepared culture medium, we observed elongated adherent cells which have undergone extensive proliferation between days 5 and 9, with colony forming around days 10 to 14. The cell population reached 70 – 80% confluence within 3 weeks in culture. Independent on the isolation procedure, the largest number of cell colonies has been obtained for a cell seeding density of 106 cells/cm2.