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Material and Methods 10

Material and Methods 10 2. Material and Methods 2.1. Mesenchymal stem cell characterisation and cell culture 2.1.1. Mesenchymal stem cell extraction Human MSCs were obtained from the femoral bone marrow of a 43 years old healthy, male donor according to the assignment of the medical faculty ethics commission (Project Number 311/04). Written consent was acquired prior obtaining of bone marrow. Furthermore, the patient was negative for HIV (human immunodeficiency virus) and hepatitis C. Mononuclear cells were separated by a Ficoll density gradient and the final centrifuged cell pellet was resuspended in full cell culture media (see below). Cell suspension was then equally divided into two portions. One half was further cultivated under standard normoxic conditions (21% O2), whereas the other half was cultured under hypoxic conditions (2% O2) directly after acquisition. Media of plastic adherent cells was changed every 3-4 days for 14 days until an appropriate amount of cell colonies were observable and splitted subsequently to passage 1. 2.1.2. Cell culture conditions Cells were cultured in minimum essential medium (α-MEM) GlutaMAX TM culture media (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS, Sigma-Alrich, St. Louis, MO, USA) and 40 IU/ml penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria) either in a normoxic (21% O2) humidified incubator (Hera cell 240, Thermo Scientifc, Schwerte, Germany) or in a hypoxic (2% O2) humidified incubator (MCO-5M, Sanyo, München, Germany) both at 5% CO2 and 37°C in a T-225 cell culture flask (NUNC, Langenselbold, Germany). Medium was changed every 3 days and cell confluency of 50% was never exceeded to prevent differentiation 68 . Trypsinization was performed after washing the cells twice with phosphate-buffered saline without Ca 2+ & Mg 2+ (PBS, PAA Laboratories GmbH, Pasching, Austria) using 0.5 g/l Trypsin (Invitrogen, Karlsruhe, Germany) with 0.2 g/l ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA-Na, Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS in a humidified incubator at 5% CO2 and 37°C

Material and Methods 11 until cells were detached. Reaction was stopped with double volume of complete culture medium and cells were replated. All cells were tested regularly for mycoplasma contamination with polymerase chain reaction (Venor ® GeM Mycoplasma Detection Kit, minerva-biolabs, Berlin, Germany). 2.1.3. Osteogenic differentiation To ensure the stem cell character of the obtained cells, 3,000 cells/cm 2 in passage 2 were plated in a 6-well cell culture dish (NUNC, Langenselbold, Germany) and expanded to approximately 90% confluence. Osteogenic induction was started with D-MEM high glucose media (PAA Laboratories GmbH, Pasching, Austria), 10% FBS, 40 IU/ml penicillin/streptomycin, 100 nM dexamethason, 10 mM β-glycerophosphat and 50 µM L-ascorbic acid (all from Sigma-Aldrich, Deisenhofen, Germany), cells with normal culture media (α-MEM) without osteogenic supplements served as a control. Media was changed twice a week for 21 days. Experiments were performed twice in triplicates. At the end of the osteogenic differentiation, cells were washed twice with PBS (with Ca 2+ - & Mg 2+ -ions), fixed with 4% paraformaldehyd (PFA, MicroCos GmbH, Garching, Germany) for 10 minutes at room temperature (RT) and incubated with Alizarin Red Stain Solution (Osteogenesis Assay Kit, Millipore, Billerica, USA) for 20 min at RT. Subsequently cells were washed three times with distilled water and samples were air-dried. Histologic images were acquired with a Axiovert 40 CFL microscope (Carl Zeiss, Göttingen, Germany) equipped with a RGB colour camera (Axiocam ICc3, Carl Zeiss, Göttingen, Germany) and a 10x magnification phase contrast objective (A-Plan, Carl Zeiss, Göttingen, Germany). Alizarin Red quantification was performed according to the company’s protocol. In brief, stained cell monolayer was incubated with 10% acetic acid for 30 min at RT and afterwards mechanically detached with a cell scraper and heated in microtubes for 10 min at 85°C. Following, optical density was measured at 405 nm in an ELISA reader (Multiskan FC, Thermo Scientific, Schwerte, Germany) and all sample values were compared, subsequent background subtraction, to a standard curve for absolute Alizarin Red content.

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