P-EMBL-165 - P-EMBL-165
In this study we analyzed mesenchymal stem cells (MSC) from the bone marrow (BM) that were isolated form the same donors under two different growth conditions (M1 and M2). Bone marrow aspirates were obtained from the iliac crest of four healthy donors aged 25-35 years after approval by the Heidelberg University Ethical Board (approval nos.: 042/2000 and 251/2002). About 10-30 ml bone marrow aspirate were collected in a syringe containing 10,000 IU heparin to prevent coagulation. The mononuclear cell fraction was isolated by Biocoll density gradient centifugation (d=1.077 g/cm3; Biochrom, Germany).
BM-MSC-M1 were cultivated as published before (Reyes et al. 2001). In brief, mononuclear cells were plated in expansion medium (M1) at a density of 100,000 cells/cm2 in tissue culture flasks (Nunc) coated with 10 ng/ml fibronectin (Sigma). The expansion medium consists of 58% Dulbecco's Modified Eagles Medium-Low Glucose (DMEM-LG, Cambrex) and 40% MCDB201 (Sigma), 2% FCS (Stemcell Technologies), supplemented with 2 mM L-Glutamine, 100 U/ml Pen/Strep (Gibco), 1% insulin transferrin selenium, 1% linoleic acid bovine serum albumin, 20 nM dexamethasone, 0.1 mM L-ascorbic-acid-2-phosphate (all from Sigma), PDGF-bb and EGF (10ng/ml each, R&D Systems). On reaching 80% confluency, cells were trypsinized with 0.25% trypsin /1ml EDTA (Invitrogen) and replated at 2,000-10,000 cells/cm2. Cells were expanded for 2-6 passages. Additionally, cells were successfully differentiated into bone, cartilage and fat following the standard protocols (Pittenger et al, 1999).
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