P-EMBL-168 - P-EMBL-168
Mesenchymal stem cells (MSC) form human cord blood (CB) were isolated as follows: Human umbilical cord blood (CB) was collected after informed consent of the mother by a standardized procedure using syringes containing L-Heparin as anticoagulant. After 2:1 dilution with PBS, MNC were obtained by Ficoll density gradient centrifugation (400g for 25 min). The cells were washed twice in PBS und seeded at a density of 1-3x106 cells/cm2. Growth of adherent cells was initiated in myelocult medium (StemCell Technologies) with dexamethasone (10-7 M; Sigma-Aldrich), penicillin (100 U/ml; Gruenenthal), streptomycin (0.1 mg/ml; Hefa-pharma) and ultraglutamine (2 mM; Cambrex) as previously described by Koegler et al., 2004. Non adherent cells were removed after 72 h, and the adherent cells of fibroblastoid morphology were fed weekly with culture medium. Expansion of the cells was performed in Mesencult basal medium with additive stimulatory supplements according to the manufacturer's instructions (M3; StemCell Technologies). CB-MSC-M3 were subjected to differentiation assays and flow cytometric analysis. Osteogenic in vitro differentiation assays were successfully performed as described before by Pittenger et al., 1999.
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