P-EMBL-167 - P-EMBL-167
Human adipose tissue (AT) of four healthy donors aged 21-40 years was obtained from elective liposuction procedures under anaesthesia after informed consent using guidelines approved by the Ethic Committee on the Use of Human Subjects (Cytonet). Mesenchymal stem cells (AT-MSC-M1) were isolated as described before (Hauner et al., 1989). In brief lipoaspirates were washed with sterile phosphate-buffered saline (PBS). A two step digest in Krebs-Ringer (pH 7,4) buffered with 25 mM Hepes containing 20 mg/ml BSA and 1,5 mg/ml collagenase (CLS type I) was performed for 30 min at 37C under permanent shaking, followed by filtration through a 149 um mesh filter. Cell suspensions were centrifuged at 200g for 10 min, and contaminating erythrocytes were removed by erythrocyte lyses buffer pH 7,3. After washing filtrate cells were cultivated in the same expansion medium M1 as used for BM-MSC-M1 (Reyes et al., 2001). This 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). Cell populations were highly homogenous in morphology. Osteogenic and Adipogenic differentiation was successfully induced under appropriate in vitro culturing conditions as described before (Pittenger et al., 1999).
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